Note: Descriptions are shown in the official language in which they were submitted.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
1
Plant pathogen inducible control sequences operably linked to cell cycle
genes and the uses thereof
The present invention is generally directed to plant pathogen inducible
control
sequences such as promoters which are operably linked to cell cycle genes and
which are - in combination - capable of modifying the cell cycle of a plant
cell thereby
conferring disease resistance in transgenic plants.
Summary of the invention
Plant pathogens cause a multitude of diseases of great economic impact for
many
agriculturally significant crop plants such as potato, tomato, soy bean,
sugarbeet,
maize, wheat, rice, barley, vegetables and oilseed rape to name a few.
Relevant
pathogens include, nematodes, viruses, viroids, fungi, bacteria and insects. A
variety
of resistance strategies are employed to combat infection and disease ranging
from
chemicals, biological control, crop rotations, traditional breeding and more
recently,
genetic engineering through the introduction into the plant of resistance
genes, toxin
genes and plant defense genes; see the following general reviews for the
current
state-of-the-art with respect to genetic engineering of pathogen resistant
transgenic
plants: nematodes: Jung (1998); insects: Schuler (1998); bacteria: Mourgues
(1998).
The approach for plant protection against pathogens that is presented by this
invention differs significantly from the genetic resistance strategies that
form part of
the state-of-the-art. Most of the current strategies are based on the cloning
of natural
resistance genes (mostly encoding pathogen-recognition proteins) or on the
engineering of proteins with anti-pathogen activity. Their starting point is
molecular
plant pathology research, in particular the study of proteins that are part of
the host's
defense against pathogens. Their other starting point which uses the selective
ablation or "suicide" of infected cells through the use of cytotoxins (e.g.
barnase/barstar) strictly relies on having promoters that are not leaky in
order to
avoid severe side effects from unwanted cell death. The inventive strategy
that is
proposed herein comes from a different angle and proposes a solution to these
problems. The invention is therefore the conferring of pathogen disease
resistance
by selectively (through the use of pathogen inducible promoters) modifying the
cell
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
2
cycle of the plant (through for instance, arresting the cell cycle) which is
activated
and/or commandeered in response to pathogens.
The advantages of the strategy taught by the current invention are
multifarious. First,
inhibiting the cell cycle upon pathogenic infection is a non-destructive
method and
will not affect the physiology of for instance the root system upon nematode
infection. Second as the expression of genes which arrest the cell cycle, have
no
effect on non-dividing cells, the pathogen inducible promoters can be leaky in
non-
dividing cells. Third, the strategy should also give both broad-range and long-
term
resistance against many species of pathogens, as opposed to strategies that
are
based on plant receptor proteins for recognition of specific races of
pathogens.
Fourth, use of plant genes as opposed to the introduction of DNA from bacteria
or
other non-plant organisms (e.g. Barnase) is preferable from a regulatory and
consumer acceptance point of view. Fifth, cell cycle proteins function in the
complex
context of the plant cell cycle machinery. Horizontal transfer of these genes
into
microbial soil organisms should not give any selective advantage to the
latter. Sixth,
successful pathogen resistance through transgenic cell cycle technologies will
substantially reduce the use of highly toxic pesticides.
The inventors and others have shown that many plant pathogens affect the state
of,
or rely on, the cell cycle in infected host cells for their growth,
replication or
reproduction. The specific changes to the cell cycle depend upon the pathogen
in
question. This invention provides a method for targeting through the use of
pathogen
inducible promoters in combination with cell cycle control proteins, the
arrest or
regularization of the cell cycle in infected cells which inhibit growth,
replication or
reproduction of the pathogens involved.
The invention therefore involves the inventive combination of two essential
components, cell cycle control proteins and pathogen inducible promoters to
confer
disease resistance in plants.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
3
Background of the invention
Several documents are cited throughout the text of this specification. Each of
the
documents cited herein (including any manufacturer's specifications,
instructions, etc.)
are hereby incorporated by reference; however, there is no admission that any
document cited is indeed prior art as to the present invention.
Nematodes
Plant parasitic nematodes are pathogens that infect a wide range of
economically
important plant crops causing severe losses to agriculture that can amount to
more
that US$100 billion per year world wide (Opperman and Bird, 1998; Sasser and
Freckman, 1987; AgBiotech News and Information 10, p. 12, 1998). In the U.S.
it is
estimated by the U.S. Society of Nematologists that nematodes cause an average
12% loss in overall productivity, amounting to a loss of 6-8 billion US$.
Yield losses
pose a major economic problem. In potatoes for instance, the estimated annual
losses due to potato cyst nematodes in Europe amount to over US$480 million
(Agrow, 280, p. 21, 1997). Cyst nematodes are the most economically damaging
disease to soybean with annual losses in the North Central region of US
amounting
to US$267 million (http://ianrwww.unl.edu/ianr.plntpath/nematode/son/nn nema
htm)
In cereal crops such as wheat and barley, losses can attain or exceed 50% when
nematode proliferation coincides with the development of young crops. This is
so for
Australian and Indian wheat crops, for hardwheat in France and spring cereals
in
northern Europe. in Australia, where H. avenge damage to wheat is the highest
recorded, costs of treatment have been estimated at AU$72 million per annum
for 2
million ha infected area. Cyst and root-knot nematodes also pose a major
problem in
cultures such as sugarbeet, coffee, cacao and fruits such as bananas. Root-
knot
nematodes are important pests in many vegetable crops, particularly in warmer
areas. Yield losses up to 50% have been reported in tomato in Southern Italy
and
other parts of the Mediterranean region. In South Africa the average yield
loss in
tomato due to nematodes is estimated at 20.6%.
With such a serious impact on yield and production it is therefore important
to
develop effective methods of nematode control. Existing methods include: crop
rotation, chemical nematicides, traditional breeding, and genetic engineering
of
resistance. Crop rotation is the most simple mechanism to restrict yield
losses by
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
4
nematodes. However, this method has a number of practical drawbacks. Firstly,
farmers are often reluctant to introduce crop rotation or to keep to the
recommended
rotation scheme. Secondly, crop rotation requires knowledge of the nematodes
that
are present in the soil. This information is often either not available or
incomplete.
For example, low-abundant species or pathotypes are easily overlooked in a
mixed
population with standard methods, while the technologies to fully identify a
mixture of
different nematode species/pathotypes are too expensive to be used at a large
scale. Moreover, certain nematodes persist for very long times in soil and are
easily
reintroduced, for example through green fertilization. Another important
problem is
that some nematode species have such a broad host range that crop rotation is
not
feasible due to the lack of resistant plant species/cultivars. This is for
example the
case for Meloidogyne chitwoodi, which poses an increasing problem to European
agriculture since 1980. A similar problem may occur with another species with
broad
host range, Meloidogyne faliax, which has been reported in Belgium for the
first time
in 1996.
The major means of nematode control has been the application of chemical
nematicides. Yet, with the nematicide market standing at only $700
million/year
(against $100 billion/year crop losses due to nematode parasites), the
inadequacy of
nematicide strategies is obvious. Nematicides are generally highly toxic
compounds
known to cause substantial environmental impact. In the past several years,
issues
such as ground water contamination, mammalian and avian toxicity, and residues
in
food have caused much tighter restrictions on the use of nematicides. fn many
countries, the concentrations of nematicides that are permitted during potato
cultivation are so low that they are not effective, implying that nematicides
are
essentially not an option anymore for potato cultivation. Nematicides are
still allowed
during sugar beet cultivation, but only at the time of sowing. As a
consequence,
nematode infestations are still encountered later during the growing season.
In
practice, because of the lack of alternatives, growers still apply nematicides
at a
large scale, in spite of current regulations.
Some successes have come forward from breeding programmes. Genetic resistance
to certain nematodes is available in some cultivars, but these are restricted
in
number and the availability of cultivars with both desirable agronomic traits
and
nematode resistance is limited. For example, popular potato cultivars that are
grown
for consumption (such as Bintje and Desiree) are sensitive to a broad range of
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 nematodes, while nematode-resistant potato varieties lack the desired traits
of Bintje
or Desiree. In addition, traditional plant breeding is a slow process,
requiring
generally 5-10 years for the production of a new cultivar.
With the development of marker-assisted breeding and gene technology, one may
expect that the time required for developing a new variety will shorten
significantly in
the future. Some resistance genes have been mapped, for example, in potato:
Groi
(Ballvora et al., 1995; Jacobs et al., 1996), and H1 (Niewohner et al., 1995);
in
tomato: Mid (Yagoobi et al., 1995) and Hero (Ganal et al., 1995) and Mi-t
(Ganal
and Tanksley, i 996); and Hs 1p~°-' of sugar beet (Cai et al., 1997);
see also Jung
(1998) and references cited therein for further examples of nematode
resistance
genes. Programmes for the introduction of these genes in sensitive varieties
have
been initiated. However, strategies based on breeding or genetic engineering
of
resistance genes have some inherent weaknesses. Firstly, resistance genes
usually
operate against a very limited number of nematode races. Secondly, resistance
mechanisms based on single resistance-genes are rapidly broken because of the
very strong selection pressure in modern agriculture (genetically uniform
plant
populations) and the monogenic basis of the resistance trait {often a gene-for-
gene
recognition event).
Alternative molecular strategies are being developed that should lead to a
more
durable and broad range resistance against plant-parasitic nematodes. These
strategies are based on the expression of proteins in plants that have a
nematicidal
impact, usually due to interference with an essential metabolic or structural
process
in the nematode. Examples include proteinase inhibitors (intertering with the
nematode's dietary uptake of proteins), lectins, chitinases, collagenases, and
Bt-
toxins (reviewed in Jung et al., 1998 and incorporated herein by reference).
The
advantage of these approaches is that they are generally not toxic to the
plant and
that these proteins therefore do not need to be expressed under control of a
highly
specific promoter. Yet, it remains to be proven whether these proteins, when
expressed in transgenic plants, have sufficient impact to completely block the
development and reproduction of the nematode. A preliminary study with
transgenic
tomato plants expressing a Bt-toxin showed a reduction in eggmass of
Meloidogyne
of about 50% (Burrows and de Waele, 1997), but further studies and field
trials have
to be performed to corroborate the impact of Bt-toxin on plant-pathogenic
nematodes. Since many of these proteins have one or a few target
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
6
proteins/structures in the nematode, resistance may still be broken rather
rapidly
under conditions of strong selective pressure, similarly as in the case of the
monogenic resistance traits; see above.
Another approach consists of engineering a suicide-construct that is activated
upon
infection (or feeding initiation in the case of sedentary nematodes), thereby
killing the
invader. Essential to the success of such an approach is the availability of a
tightly
controlled promoter. This problem can partially be circumvented by using a two-
component system, consisting of a toxic protein for suicide and a second
detoxifying
protein for backing-up promoter leakiness in specific tissues or upon specific
environmental conditions. Although such systems have been developed (e.g.
barnase and barstar, Strittmatter et al., Bio/Technology 13 (1995), 1085-1089)
no
commercialization has been pursued so far, due to recurrent problems with
uncontrolled expression of the highly toxic protein.
Several plant parasitic nematode genera have evolved the ability to induce
morphological changes in host cells to form feeding sites. The classification
of these
root parasites and their life cycles has been reviewed in Sijmons (1994). The
cytological and especially nuclear changes that are induced by two main groups
of
sedentary endoparasites: root knot and cyst nematodes, has been reviewed by
Gheysen (1997). These nematodes are the most damaging species on a wide range
of host plants (over 2000 for Meioidogyne) and are therefore a very important
pest in
agriculture. After an initial invasion and migration towards a suitable site
in the plant
root, these nematodes become immobile and completely depend on the successful
induction and maintenance of specialized feeding cells. These parasites are
therefore biotrophic; they do not kill the cells they feed from but instead
modify them
into efficient food sources, most probably by the injection of unknown
substances
originating from their oesophageal glands (Hussey, 1989). Also some
ectoparasitic
nematodes such as Xiphinema species form galls on the roots of their hosts.
The
mechanism of feeding site formation is different and specific for the
infecting
nematode, regardless of the tissue and host in which they are induced. Root
knot
nematodes induce several giant cells embedded in a gall while cyst nematodes
generate a syncytium. However, the final large and multinucleate feeding cells
are
functionally similar, in that they are metabolically highly active and adapted
to
withdraw large amounts of nutrient solutions from the vascular system of the
host
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
7
plant in order to feed the nematode. This functional analogy is reflected in
the
ultrastructure of the feeding cells: cell wall ingrowths adjacent to vascular
tissue,
breakdown of the large vacuole, dense granular cytoplasm with many organelles
and
numerous enlarged amoeboid nuclei (Bird, 1961; Jones and Northcote, 1972;
Jones,
1981 ). In fact, the induction of cell cycle gene expression is one of the
first events
during the initiation of both types of feeding cells, giant cells as well as
syncytia
(Niebel et al., 1996).
The response of host cells to cyst nematode infection is the formation of a
syncytium, a large multinucleated hypertrophied cell generated by the fusion
of
i 5 neighboring protoplasts after partial cell wall dissolution. In sharp
contrast to giant
cells, convincing evidence for cell wall breakdown was obtained for syncytia
induced
in many host plants by different cyst nematodes (Endo, 1964; Jones and
Northcote,
1972; Jones, 1981; Magnusson and Golinowski, 1991 ). The enlargement of nuclei
indicates that DNA multiplication is taking place within the syncytial tissue
during and
after the incorporation of new cells through cell wall dissolution (Endo,
1964). To get
insight in the location and timing of DNA synthesis in syncytia, 3H-thymidine
incorporation experiments were done in soybean roots infected by Heterodera
glycines (Endo, 1971 ). Moderate levels of 3H-thymidine were incorporated in
nuclei
of syncytia up to 16 days after inoculation, indicating that syncytia are
relatively
quiescent in terms of DNA synthesis. However, a high 3H-thymidine labeling was
observed at the borders of syncytial cytoplasm with adjacent normal root
tissue, and
even later than 16 dpi, albeit at a lower level, label was still apparent in
this contact
zone. At this leading edge, preparation of cells for incorporation seems to
involve
DNA synthesis. This could mean that cells adjacent to the syncytium are
activated
for mitosis and the stimulated nuclei might be drawn into the syncytial
cytoplasm
before or after karyokinesis or cytokinesis (Endo, 1971; Magnusson and
Golinowski,
1991).
Although there were many early reports that cell wall breakdown and fusion of
neighboring cells contribute to their formation (Dropkin and Nelson, 1960;
Bird, 1961;
Rohde and McClure, 1975), it is now generally believed that giant cells
develop by
repeated mitosis without cytokinesis (Huang and Maggenti, 1969 a & b; Jones
and
Northcote, 1972; Jones and Payne, 1978; Jones, 1981 ). Advanced EM techniques
could not demonstrate cell wall dissolution in developing giant cells, despite
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
8
extensive searches. Furthermore, Jones and Payne (1978) showed that cell plate
vesicles initially lined up between the two daughter nuclei but then
dispersed,
resulting in the abortion of the new cell plate formation. Although giant
cells with as
many as 150 nuclei have been reported in Glycine max (Dropkin and Nelson,
1960),
the mean number of nuclei per mature giant cell is between 30 and 60 in most
studied plant hosts (Starr, 1993). The increase rate of the number of nuclei
for all
studied plant species is greatest during the first 7 days after inoculation
and no
mitotic activity was observed in giant cells associated with adult nematodes
(Starr,
1993). In pea giant cells, it was observed that the number of nuclei doubled
each day
during the period of highest mitotic activity (Starr, 1993).
By using cell cycle blockers, DNA synthesis and progression through S2 phase,
or
mitosis, have been shown to be essential for plant cells to develop into gall
and
syncytium establishment. The herbicide oryzalin inhibits plant microtubule
polymerization and arrests cells at the early M phase (Morejohn et al., 1987),
whereas
hydroxyurea is a cytostatic drug acting as a specific inhibitor of DNA
synthesis (Young
and Hodas, 1964). Control experiments showed that high concentrations of
hydroxyurea or oryzalin were not harmful for the nematodes themselves.
Upon hydroxyurea treatment, early giant cell and syncytium development was
blocked
in Arabidopsis (de Almeida Engler et al., 1999). This demonstrates that genome
multiplication is essential for the formation of both types of feeding cells.
Application of
hydroxyurea at later stages resulted in normal development of the nematodes.
Upon oryzalin application (dpi = 1 and 3), root knot nematode development in
Arabidopsis was completely inhibited. The formation of giant cells was
initiated but their
development was severely hampered. Moreover, they contained a reduced number
of
nuclei as compared to untreated giant cells. When oryzalin was applied at
later stages
(dpi = 9), the majority of the nematodes were able to complete their life
cycle. This is
consistent with the fact that after 9 dpi no nuclear division occurs and that
mitosis is
required only for early giant cell differentiation. If mitosis was not
involved in syncytium
formation, oryzalin should not affect cyst nematode development. Application
of oryzalin
at dpi = 1 resulted in the complete inhibition of syncytium development and no
cysts
were formed on these plants. When oryzalin was applied at later stages (dpi =
3 and 9)
an increasing number of the infective juveniles developed into cysts. These
data
support the notion that mitotic activity is required for proper syncytium
development. It
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
9
was observed that oryzalin inhibits the mitotic activity in cells prior to
syncytium
incorporation and as a consequence, syncytium expansion is restricted.
Other examples of nematodes that interact with the cell cycle are listed in
Table 1.
Accordingly, alternative approaches for genetic engineering of artificial
nematode
resistance had been needed. Hence, the present invention surprisingly
succeeded in
providing transgenic plants which are resistant to a broad range of nematode
species with an approach that is safe for the host plant and the environment.
Geminiviruses
Viruses are the causative agents of a large number of serious and potentially
serious
diseases in humans, animals and plants. Plant viruses in particular have the
potential to destroy or reduce crop yield and to otherwise have a deleterious
effect
on agricultural and horticultural industries to economically significant
levels.
Particularly important viruses in this regard are the DNA viruses, including
the
geminiviruses.
The geminiviruses are a large and diverse family of plant viruses comprising
three
genera, Mastre-, Curto- and Begomoviruses. Classification is based on genome
structure (mono- or bipartite), natural vector (leafhoppers or white fly
species) and
host range (mono or dicotyledonous). Mastrevirus are transmitted by
leafhoppers
and except for a few exceptions infect monocots; their genome comprises a
single
stranded DNA component. Most Begomoviruses are transmitted by white fly
species,
infect dicots and posses a bipartite genome, usually called A and B, of
similar sizes.
Curtoviruses occupy an intermediate position infecting dicots but with a
single
stranded DNA genome component. This classification is in accordance with the
phylogenetic groups obtained in evolutionary studies.
Examples of Mastrevirus include: Maize Streak Virus (MSV), Digitaria Streak
Virus
(DSV) and Wheat Dwarf Virus (WDV). Examples of Curtovirus include: Beet Curly
Top Virus (BCTV) and Horseradish Curly Top Virus {HCTV). Examples of
Begomovirus include: Bean Golden Mosaic Virus (BGMV), Texas Pepper
Geminivirus (TPGV), Squash Leaf Curl Virus {SqLCV), Abutilon Mosaic Virus
{AbMV), Ageraturn Yellow Mosaic Virus (AYMV), African Cassava Mosaic Virus
(ACMV), Chloris Striate Mosaic Virus (CSMV), Tomato Yellow Leaf Curl Virus
(TYLCV), Tomato Golden Mosaic Virus (TGMV) and Tomato Leaf Curl Virus {TLCV).
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 There are many other examples of geminiviruses that can be identified by
persons
skilled in the art (see for examples the online viral database VIDE Database
(at ANU
Bioinformatics Group) at http://bioloay.anu.edu.au/Groups/MES/vide/genus005
htm)
and that are known to cause economically important diseases affecting yield
and
quality of crops.
10 The geminiviruses are a group of small DNA viruses which infect plant
cells. They
contain a single strand of circular DNA referred to as "virion-sense" or
"positive-
sense" DNA of less than about 2900 base pairs. Upon infection of a host cell
by a
geminivirus, the viral coat protein is removed and a double stranded
replicative form
of DNA is synthesized comprising the virion-sense strand and a "complementary-
sense" strand. Transcription occurs from both the virion-sense strand and from
the
"complementary sense" strand, giving rise to (+) and (-) sense RNA transcripts
respectively.
Geminiviruses replicate in the nucleus of the infected cell and are thought to
employ
a rolling-circle mechanism similar to one used by the single-stranded DNA
containing coliphages and certain Staphylococcus aureus and Bacillus subtilis
plasmids. The other known plant viruses, including other plant DNA viruses,
replicate
via RNA intermediates.
Geminivirus particles accumulate in the nuclei of infected cells where DNA
replication and virus assembly probably take place (Davies et al., 1987).
Their
putative replicative forms are double-stranded covalently closed circular DNA
of
about 2,7 Kb in chromatine-like structures and are likely to be the
transcriptionally
active forms of the virus (Abouzid et al., 1988).
As mentioned above, geminiviruses cause important diseases in a number of
crops.
No effective control strategy has been developed to date. Due to the economic
importance of plant DNA viruses and, in particular, geminiviruses, there is a
need for
disease resistance strategies to be developed. Several genetic engineering
strategies against viruses have been used based on coat protein expression,
however these approaches are not effective against geminiviruses. Other
pathogen
derived or alternative transgenic resistance strategies (such as expression of
toxic
genes - Hong et al., 1996) have been explored. Many problems have been
encountered such as low level of resistance (Day et al., 1991 ), and narrow
range
where resistance was only effective against a few strains of a virus
(Frischmuth and
Stanley, 1998; Noris, Accotto et al., 1996). Conventional plant breeding
programs
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
11
have provided partial answers in a number of cases, however, frequently its
successes are limited, primarily because natural resistance to geminivirus is
usually
poligenic and most cultivars of a certain crop remain susceptible (Hahn et
al., 1980).
The potential for an effective control strategy based on a transgenic approach
remains very high. However, there has been very little success in obtaining
transgenic plants with an enhanced tolerance to geminiviral infection. It is
therefore
an object of the current invention to propose a solution for this problem.
Fun i
Fungi also induce DNA replication and cell division in plants. Non exhaustive
examples of such fungi are described below.
Clubroot is a serious disease of crucifers caused by the primitive fungus
Plasmodiophora brassicae. The fungus penetrates the roots and induces the
continued division and enlargement of root cells. Galls can range in size from
tiny
nodules to large, club-shaped outgrowths that may involve most of the root
system
including the underground stem. Severely affected plants are stunted and wilt
under
moisture stress. Affected crucifers include canola, cabbage and cauliflower.
Infested
fields must be kept free of susceptible crops for many years because of the
long-
lived resting spores (Braselton, 1995).
A number of fungi are known to induce the formation of "brooms", i.e. a bunch
of
newly formed, swollen spongy shoots with few or no leaves. The accelerated
growth
of the broom consumes much of the plant's energy resulting in the production
of
fewer or no pods or fruits. Development of the brooms follows infection of
terminal
and auxiliary bud rneristems (vegetative brooms) or of flower cushions.
Flowers or
pods can also be infected. Examples of fungi causing witches' broom are:
CrinipeUis perniciosa (Basidiomycetes). Its host is the cocoa plant (Theobroma
cacao). The primary phase of the fungus (biotrophic/homokaryotic) initiates
the
infection and causes the broom to develop. After 6-9 weeks, the green brooms
start
to necrotize. The change is associated with dikaryotization of the fungus to a
secondary phase of the fungus (saprotrophic/dikaryotic). Subsequently,
basiodiocarps are formed on the dead brooms (Cane et al., 1982; Wheeler,
1985). C.
perniciosa is indigenous to the Amazon but has now spread into most of the
cocoa
growing regions in South America and several Caribbean islands. Losses from
witches' broom may be more than 90%. Yields in Bahia decreased by 60% from
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
12
1990 to 1994. C. perniciosa also caused a devastation of Brazil's cocoa crop.
Protective treatment with chemical fungicides is costly and usually
ineffective
(Evans, 1980; Pereira et al., 1996; Rudgard et al., 1986). Pathotypes of C.
perniciosa capable of attacking solanaceous species (e.g. potato) have been
described as well (Bastos, 1985).
Pucciniastrum goeppertianum (Basidiomyctes). Its host is lowbush blueberry
(Vaccinium angustifolium). P. goeppertianum is a relatively minor disease
(2.2% of
plants are infected) but infected plants usually do not produce fruit.
Taphrina wiesneri (Ascomycetes). Cherry (Prunus) species serve as the host.
Members of the true smut fungi (Ustilagomycetes belonging to the
Basidiomycetes)
can induce morphological gall-like distortions of different organs and in
different host
plants. The best known is Ustilago maydis. This fungus displays dimorphic
growth
switching from budding to filamentous growth. Only in its dikaryotic
filamentous
stage, U. maydis behaves as a pathogen on corn (Zea) species (Banuett, 1992).
Infected tissues, usually the ears (but also leaves and tassels), transform
into
tumorous galls. Generally, 2-5% of the plants in a corn field are infected by
U.
maydis but if the conditions are good for the smut fungus up to 80% of a field
can be
infected. The galls of U. maydis are on the other hand considered a food
delicacy. In
Mexico, they are known as "Huitlacoche" and in the USA as "maize mushroom",
"Mexican truffles" or "caviar azteca" (Valverde et al., 1995). Controls have
generally
been unsatisfactory. Some other gall-inducing Ustilagomycetes and their hosts)
are
listed below:
- Exobasidium vaccinii causing leaf galls on azalea, rhododendron and
lingonberry. Usually a cosmetic disease but it can reduce the ornamental
qualities of or the fruit production by infected plants.
- Exobasidium camelliae causing leaf galls on tea (Camellia species).
- Entorrhiza casparyana causing root galls on jointed rush.
The black knot disease is characterized by the occurrence of black warty knots
on
branches of trees infected with the fungus Apiosporina morbosum. Such trees
grow
poorly and gradually become stunted and can ultimately die. Various species
(cherries, plums, prunes, flowering almond, apricot) are reported to be
susceptible to
black knot. Combating black knot disease in susceptible varieties is difficult
and
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
13
consists of fungicide application in combination with a sanitation program
(Ogawa et
al., 1995). In all cases, the newly and aberrantly formed host tissues
ultimately
sustain the formation of spores by the fungi. Where studied, the neoplastic or
hyperplastic disease conditions caused by fungi seem to be the result of
increased
cytokinin levels.
Brassica campestris (turnip) clubs caused by P. brassicae contain amounts of
bound
and free cytokinins (zeatin and zeatin riboside) that are two to three times
higher
than in healthy turnip roots (Dekhuijzen, 1980). Furthermore, turnip explants
infected
with P. brassicae are independent of cytokinins for continued growth
(Dekhuijzen
and Overeem, 1971 ). The origin, plant-borne or released by the fungus, of the
additional cytokinins remains, however, unsolved (Dekhuijzen, 1980).
C. perniciosa exert its effect in the shoot apex near the area of initial
tissue
differentiation by causing cell enlargement and differentiation without
destroying the
basic pattern of tissue organization. The type of distorted growth (loss of
apical
dominance resulting in the broom) might suggest an alteration in growth
regulator
balances. Diseased tissue was indeed found to contain very small although
significant increases of the cytokinin zeatin riboside relative to healthy
tissue
(Orchard et al., 1994). Cocoa cell suspensions responded to primary phase C.
perniciosa mycelium by doubling growth which was stopped and declined by the
appearance of secondary phase mycelium (Muse et al., 1996).
The effects of cytokinin on the plant cell cycle are well documented. Cell
division is
induced by cytokinin and this effect is mediated through elevated levels of
cyclin D3.
Constitutive expression of cyclin D3 in transgenic plants allowed induction
and
maintenance of cell division in the absence of exogenous cytokinin (Riou-
Khamlichi
et al., 1999; Soni et al., 1995). Plant cyclin D3 controls Go-G,progression
but might
also be involved in G2-M transition (for review, see Mironov et al., 1999).
Cytokinins
also affect G2-M transition through cdc25. Cell division in Nicotiana
plumbaginifoiia
expressing the cdc25 gene from fission yeast proceeds independent of cytokinin
(John, 1998).
A transgenic approach has been used already successfully to enhance resistance
of
Arabidopsis against the clubroot pathogen P. brassicae. The method consists of
constitutively expressing viscotoxin, a toxic thionin from the mistletoe
Viscum album,
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
14
in all organs of Arabidopsis (Holtorf et al., 1998). An alternative and novel
approach
suggested by this invention would be to inhibit the formation of the clubs by
blocking
the host root cell cycle. As such, the formation of the tissue necessary for
the fungus
to produce spores is prevented.
Other pathogens
Nematodes, geminiviruses and fungi are not the only pathogens that influence
the
cell cycle in plants. An example of a insect pathogen includes the highly
polytene
cells in galls induced by the midge Mayetiola poae on stems of the grass Poa
memoralis (Hesse, 19fi9). In other insect induced galls multinucleate cells
are
formed by acytokinetic and other polyploidizing mitoses (Hesse, 1971;
Shorthouse
and Rohfritsch, 1992).
Other viruses which cause a cell proliferation in plants upon infection belong
to the
class of Reoviridae. Examples of these viruses, which can infect plant cells
are
Fijivirus, Phytovirus and Oryzavirus. This class of viruses also embraces
viruses of
vertebrates and invertebrates. The majority of these viruses has a limited
host range
and is for instance restricted to the Gramineae. However the wound tumor virus
has
a broad host range in dicotyledonous plants. The viruses concerned replicate
in
phloem cells and cause proliferation of phloem cells and as a consequence
thereof
the formation occurs of galls and tumors in leaf, vein, stem and root.
Other viruses that would be relevant to the present invention include the
Nanoviruses
-examples include the milk vetch dwarf virus (MDV), the banana bunchy top
virus
and the faba bean necroticyellows virus. It has been suggested that MDV
interact
with the cell cycle (Sano et al., 1998).
Several other pathogenic organisms which cause cell or tissue proliferation in
plants
are parasitic plants like Arceuthobium sp. (dwarf mistletoes) and bacteria
like
Agrobacterium tumefaciens, Rhodvcoccus fascians, Pseudomonas savasfanoi,
Xanthomonas campestris pv citri. or Erwinia herbicola.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 Description of the present invention
Resistance strategies against pathogens fall into two broad categories -namely
the
use of "resistance genes" or "toxic/suicide" genes. The classical strategy
using
resistance genes makes use of constitutive promoters to express the resistance
10 gene (e.g. pathogen recognition genes or specific toxins). However, the
strategy
usually only provides narrow resistance (against only one or a few species of
pathogen) and a resistance which is relatively easy for the pathogen to
overcome.
The other main resistance strategy uses genes that eliminate the pathogen by
provoking a suicide mechanism in the plant. In this strategy a very specific
promoter
15 must be used to ensure the timely (on pathogen infection) and tissue
specific
expression (infected cells) of the suicide toxin so as to avoid harmful side
effects to
the plant. To our knowledge a promoter that fulfils these requirements has so
far not
been found.
However, the current invention - namely the combination of a pathogen
inducible
promoter and a cell cycle gene -provides for a strategy where the promoter
need not
necessarily have a very strict activity profile and where the gene (i.e. the
cell cycle
gene) would be effective against a broad range of pathogens both in type
(fungus,
virus, nematode, etc.) and in species within that type. Because of the way in
which
cell cycle genes operate and a pathogen's use of that cell cycle (in
particular the
activation of certain cell cycle related events in cells/tissues which are the
primary
target of the pathogen), the current invention allows some ieakiness in the
promoter
such as in non-dividing cells (which are the majority of cells in a plant) or
even in
dividing cells provided those dividing cells are of no agricultural
importance. This use
of cell cycle also means that the pathogen will not be able to develop
resistance
because the host's cell cycle is an essential aspect of the pathogen's life
cycle.
Thus, the technical problem of the present invention is to provide means and
methods that can be used for engineering of broad range disease resistant
plants
taking into account ecological and economic needs.
The solution to this technical problem is achieved by providing the
embodiments
characterized in the claims.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
1 fi
Accordingly, the invention relates to a chimeric gene or recombinant DNA
molecule
comprising at least a plant pathogen inducible control sequence operably
linked to a
cell cycle gene that is preferably capable of modifying the cell cycle,
preferably
arresting the cell cycle or cell division of a plant cell. Advantageously,
said cell cycle
gene is capable of modifying the cell upon pathogen infection, preferably due
to the
induced expression triggered by the pathogen inducible control sequence, i.e.
promoter.
The terms "pathogen inducible control sequence" and "pathogen inducible
promoter"
are used interchangeable herein and mean that said control sequence and
promoter
are capable of regulating the transcriptional activation of a heterologous DNA
sequence.
The term "pathogen inducible promoter" includes a "pathogen responsive
promoter"
or a "pathogen targeted promoter".
A "pathogen responsive promoter" is a promoter which is induced or upregulated
in
response to pathogen infection and, preferably in cell/tissues which are the
primary
target of the pathogen.
A "pathogen targeted promoter" is a promoter which is active prior to
infection in
cells/tissues which are the primary target of the pathogen. An example of such
a
"pathogen target promoter" is the root cortex promoter (see, for example, the
ToRD2
promoter - WO 97/05261 ). For instance, in potato the root cortex cells are
the target
for feeding site initiation by the cyst nematode Globodera rostochiensis.
Preferably
the pathogen inducible promoter is not leaky in actively dividing cells,
however,
leakiness is permitted in dividing cells provided that the dividing cells are
of no
agricultural importance. The pathogen inducible promoter may be leaky in non-
dividing cells. Pathogen inducible promoters may be activated by different
classes of
pathogens.
A combination of both (basal expression from a pathogen targeted promoter and
high expression after infection from a pathogen-responsive promoter).
Pathogen inducible promoters can be derived and isolated from genes involved
in
compatible and incompatible interactions, including those required for the
pathogen
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
17
to complete its life cycle and those involved in defense responses either
directly or
as a secondary consequence.
Preferably said control sequence or promoter is inducible by either a virus, a
viroid, a
nematode, a fungus, a bacterium, an insect or a parasitic plant.
Examples of pathogen inducible promoters suitable for use in genetic
constructs of
the present invention include those listed in Table 2, amongst others. The
promoters
listed in Table 2 are provided for the purposes of exemplification only and
the
present invention is not to be limited by the list provided therein. Those
skilled in the
art will readily be in a position to provide additional promoters that are
useful in
performing the present invention. The promoters listed may also be modified to
provide specificity of expression as required.
Using methods known to the person skilled in the art, additional pathogen
inducible
promoters can be identified. One such method is promoter tagging as described
by
Barthels et al., 1997; Topping et al., 1991; Koncz et al., 1989; Kertbundit et
al., 1991
- where a promoter trap system consists of a collection of transgenic
Arabidopsis
plants, which contain random T-DNA insertions of a promoter-less ~i-
glucuronidase
(gus) gene. Gus expression in these Arabidopsis lines is a reflection of the
regulatory
elements that flank the gus insertion site. Activation of gus expression after
infection
with a pathogen is monitored by a histochemical enzymatic assay which is a
rapid
and sensitive method applicable to intact plants. Promoters of lines which
show a
desired expression pattern can then be cloned by inversed PCR techniques and
screening of genomic libraries.
Other collections of Arabidopsis such as that of Dr J Haseloff (MRC, Cambridge
England, Haseloff et al., 1997), and the collection of T-DNA tagged
Arabidopsis
mutants at INRA-Versailles (Dr. George Pelletier), can also be screened for
pathogen inducible promoters. Other methods for the isolation of pathogen
inducible
promoters include screening transposon tagged lines (Fitzmaurice et al., 1992;
Federoff et al., 1984) or differential screening (Gurr et al., 1991 ).
The transcriptional activation by the promoter employed in accordance with the
invention may preferably occur at the infection site but may also occur in
cells
surrounding the actual infection site, e.g., due to cell-cell interactions.
The pathogen
inducible promoter may advantageously not or only to a small extent be
inducible
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
18
upon other stimuli such as abiotic stress. Preferably, the induction from the
pathogen
inducible promoter upon pathogen infection is at least about 2-fold higher,
preferably
3-fold higher, particularly preferred 5-fold higher than its activation, if
any, by abiotic
stress.
The expression specificity conferred by the pathogen inducible promoters
employed
in accordance with the invention may not be limited to local gene expression
due to
pathogen infection, for example, they may have a basal but low expression in
the
non-dividing cells. In contrast, there is preferably no substantial expression
of
heterologous DNA sequences under the control of the pathogen inducible
promoter
of the invention in dividing tissue and/or meristematic cells in the absence
of
pathogen infection. Furthermore, the promoter may be combined with further
regulatory sequences that provide far tissue specific gene expression. The
particular
expression pattern may also depend on the plant/vector system employed.
However,
expression of heterologous DNA sequences driven by the pathogen inducible
promoters predominantly occurs upon pathogen infection.
"Cell cycle" means the cyclic biochemical and structural events associated
with
growth and with division of cells, and in particular with the regulation of
the
replication of DNA and mitosis. Cell cycle includes phases called: G0, Gap,
(G1),
DNA synthesis {S), Gape (G2), and mitosis (M). Normally these four phases
occur
sequentially however the cell cycle also includes modified cycles wherein one
or
more phases are absent resulting in modified cell cycle such as endomitosis,
acytokinesis, polyploidy, polyteny, and endoreduplication.
As used herein, the term "cell cycle control protein" shall be taken to refer
to a
peptide, polypeptide, oligopeptide, enzyme or other protein that is involved
in
controlling or regulating the cell cycle of a cell, tissue, organ or whole
organism
therein. Cell cycle control proteins and their role in regulating the cell
cycle of
eukaryotic organisms are reviewed in detail by John (1981 ) and the
contributing
papers therein; Nurse (1990); Norbury and Nurse (1992); Ormrod and Francis
(1993)
and the contributing papers therein; Francis and Halford (1995); Elledge
(1996);
Doerner et al., (1996); Francis et al., (1998); Hirt et al., (1994); and
Mironov et al.;
(1999).
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
19
Preferably, the cell cycle control protein is derived from a yeast or plant
cell or
animal cell, more preferably, from a plant cell, such as a monocotyledonous or
dicotyledonous plant cell (Mironov et al., 1999).
For the purpose of the present invention the term "cell cycle control
proteins" include
cyclins A, B, C, D and E including CYCA1;1, CYCA2;1, CYCA3;1, CYCB;1, CYCB;2,
CYC B2;2, CYCD1;1, CYCD2;1, CYCD3;1, and CYCD4;1 (Renaudin et al., 1996;
Evans et al., 1983; Swenson et al., 1986; Labbe et al., 1989; Murray et al.,
1989;
Francis et al., 1998; Dahl et al., 1995; Soni et al., 1995; Sorrell et al.,
1999) cyclin
dependent kinase inhibitor (CKI) proteins such as ICK1 (Wang et al., 1997),
FL39,
FL66, FL67 (PCT/EP98/05895), Sic1, Far1, Rum 1, p21, p27, p57, p16, p15, p18,
p19 Pines, 1995; Elledge, 1996), p14 and pl4ARF; pl3s"~' or CKSIAt (Hayles et
al.,
1986, De Veylder et al., 1997) and nim-1 (Fantes, i 979; Russell and Nurse,
1986;
1987a; 1987b); homologues of Cdc2 such as Cdc2MsB (Hirt et al., 1993); CdcMs
kinase (Bogre et al., 1997); cdc2 T14Y15 phosphatases such as Cdc25 protein
phosphatase or p80°~25 (Russell and Nurse, 1986; Kumagai and Dunphy,
1991; Bell
et al., 1993; Elledge, 1996) and Pyp3 (Elledge, 1996); cdc2 protein kinase or
p34°d°2
(Nurse and Bisset, 1981; Lee and Nurse, 1987; John et al., 1989; Feiler et
al., 1990;
Colasanti et al., 1991; Hirt et al., 1991; John et al., 1993); cdc2a protein
kinase
(Hemerly et al., 1993); cdc2 T14Y15 kinases such as weel or p107""ee'(Russell
and
Nurse, 1986, 1987a, 1987b; Elledge, 1996; Sun et al., 1999), mikl (Lundgren et
al.,
1991 ) and mytl (Elledge, 1996); cdc2 T161 kinases such as Cak and Civ
(Elledge,
1996); cdc2 T161 phosphatases such as Kap1 (Elledge, 1996); cdc28 protein
kinase
or p34~d~8 (Reed et al., 1985; Nasmyth, 1993); p40""°'S (Fesquet et
al., 1993; Poon
et al., 1993); chkl kinase (Zeng et al., 1998); cdsl kinase (Zeng et al.,
1998);
growth-associated H1 kinase (GAK) (Lake and Salzman, 1972; Langhan, 1978,
Labbe et al., 1989; Arion et al., 1988); MAP kinases described by Wilson et
al.,
(1998); Calderini et al., (1998); Binarova et al., (1998); and Bogre et al.,
(1998).
Preferred cell cycle control proteins for the present purpose of this
invention shall be
taken to include any one or more of those proteins that are involved in the
control of
entry and progression through S phase. They include, not exclusively, cell
cycle
proteins such as CDKs, CKIs, D, E and A cycfins, E2F and DP transcription
factors,
pocket proteins, CDC7/DBF4 kinase, CDC6, MCM2-7, Orc proteins, cdc45,
components of SCF ubiquitin ligase, PCNA, DNA-polymerase.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5
Other cell cycle control proteins that are involved in cycfin D-mediated entry
of cells
into G1 from GO include pRb (Xie et al., 1996; Huntley et al., 1998), E2F,
RIP,
MCM7C and potentially the pRb-like proteins p107 and p130.
10 Other cell cycle control proteins that are involved in the formation of a
pre-replicative
complex at one or more origins of replication, such as, but not limited to,
ORC,
CDC6, CDC14, RPA and MCM proteins or in the regulation of formation of this
pre-
replicative complex, such as, but not limited to, the CDC7, DBF4 and MBF
proteins.
15 For the present purpose, the term "cell cycle control protein" shall
further be taken to
include any one or more of those proteins that are involved in the turnover of
a cell
cycle control protein, or in regulating the half-life of a cell cycle control
protein, such
as, but not limited to, proteins that are involved in the proteolysis of one
or more of
the above-mentioned cell cycle control proteins. Particularly preferred
proteins which
20 are involved in the proteolysis of one or more of the above-mentioned cell
cycle
control proteins include the yeast-derived and animal-derived proteins, Skpl,
Skp2,
Rubl, Cdc20, cullins, CDC23, CDC27, CDG16, and plant-derived homologues
thereof (Cohen-Fix and Koshland, 1997; Hochstrasser, 1998; Krek, 1998;
Lisztwan,
1998; Plesse et al., 1998).
For the present purpose, the term "cell cycle control protein" shall further
be taken to
include any one or more of those proteins that are involved in the
transcriptional
regulation of cell cycle gene expression such as transcription factors and
upstream
signal proteins. Additional cell cycle control proteins are not excluded.
The present invention clearly encompasses the use of homologues, analogues or
derivatives of any of the above mentioned cell cycle control proteins which
function
in DNA synthesis, mitosis, S phase, endomitosis, acytokinesis, polyploidy,
polyteny,
and endoreduplication.
Hence, the present invention encompasses the use of cell cycle genes encoding
cell
cycle control proteins selected from the examples described above, such genes
also
including sense, antisense, dominant negative, wild-type or mutant versions
thereof
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
21
ribozymes to transcripts of cell cycle genes, antibodies to their gene
products and
any functional homologous gene related thereto. "Cell cycle genes" are genes
coding
for cell cycle control proteins naturally involved in the regulation of and/or
capable of
artificially modulating the cell cycle or a part thereof.
The term "operably linked" refers to a juxtaposition wherein the components so
described are in a relationship permitting them to function in their intended
manner.
The pathogen inducible promoter "operably linked" to a cell cycle gene is
ligated in
such a way that expression of a coding sequence is achieved under conditions
compatible with the control sequences. Expression comprises transcription of
the
cell cycle gene preferably into a translatable mRNA. Regulatory elements
ensuring
expression in eukaryotic, i.e. plant cells are well known to those skilled in
the art. In
the case of eukaryotic cells they comprise optionally poly-A signals ensuring
termination of transcription and stabilization of the transcript, for example,
those of
the 35S RNA from Cauliflower Mosaic Virus (CaMV) and the Nopaline Synthase
gene from Agrobacterium tumefaciens. Additional regulatory elements may
include
transcriptional as well as translational enhancers. A plant translational
enhancer
often used is the CAMV omega sequences, the inclusion of an intron (Intron-1
from
the Shrunken gene of maize, for example) has been shown to increase expression
levels by up to 100-fold. (Malt, Transgenic Research 6 (1997), 143-156; Ni,
Plant
Journal 7 (1995), 661-676).
In a preferred embodiment, the present invention relates to the above
described
chimeric gene and recombinant DNA molecule wherein said cell cycle gene is a
gene such as a cyclin dependent kinase gene, a cyclin dependent kinase
inhibitor
gene, a cyclin gene, a retinoblastoma gene, a cks gene, an E2F gene, a gene
encoding an upstream regulatory protein of a cyclin dependent kinase such as
cdc25, wee, nim or myt, a gene encoding a substrate for cyclin dependent
kinase, a
gene encoding a protein involved in DNA replication, endoreduplication,
karyokinesis
or mitosis or a sense, antisense, dominant negative, wild-type or mutant
versions
thereof or any fragment thereof or any functional homologous gene related
thereto.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
22
As discussed above, in the current invention pathogen inducible promoters will
be
combined with cell genes such that there is a modification of the host cell
cycle
which does not allow or inhibits pathogen growth, replication or reproduction.
Modifying the cell cycle includes modulating the cell cycle and/or one or more
phases of the cell cycle, for instance, arresting the cell cycle, inhibition
of S phase or
DNA synthesis, changing the timing of the cell cycle phases, skipping a phase
or
regularization of the cell cycle that has been manipulated by the pathogen.
For
instance with nematodes, the regularization of the nematode induced shortened
cell
cycle into normal ones would prevent the infected cell from expanding into
giant
cell/syncythis and this would be another way of depriving the nematode from
its food
source.
Numerous plant pathogens are known to affect the state of cell cycle machinery
in
host cells. A non-exhaustive list includes such diverse organisms as viruses,
nematodes and insects (see Gheysen et al., 1997, and Agrios for references).
Specific changes to the cell cycle depend on the pathogen in question.
For example, gemini-viruses are a large group of DNA containing viruses.
Replication of viral DNA occurs in the nucleus of host plant cells. Since
viral genome
does not encode genes required for DNA synthesis, viral replication must
depend
totally on the host DNA synthesis apparatus. The DNA synthesis apparatus of
the
host is therefore the primary target. There is also circumstantial evidence
for a more
general involvement of S phase enzymes and cell division during geminivirus
infection. The relevance of these processes for viral replication is yet to be
fully
elucidated but it is possible that apart from DNA synthesis components, also
other S
phase and cell cycle enzymes can be used in the resistance strategy proposed
by
this invention.
Root-knot nematodes induce in the place of infection formation of
hypertrophied
multinucleated giant cells which serve as the feeding site. Giant cells are
the result
of three cell cycle related processes:
1 ) multiple rounds of S phase and mitosis without cell division resulting in
cells
with multiple nuclei
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
23
2) polyploidization of individual nuclei as a result of endomitosis (S phase
and
mitosis without nuclear envelope breakdown)
3) endoreduplication - S phase without mitosis.
Even though there are numerous mitotic events in the giant cell, S phase is a
preferred target because in the absence of DNA synthesis mitosis will be
prevented
anyway, whereas mitotic block can be often followed by endoreduplication.
In contrast the feeding sites of cyst nematodes, syncitia, are formed via
fusion of
neighboring cells at the site of infection. However there are cell cycle
events
associated with cyst formation as well:
1 ) DNA synthesis in the syncitium probably through both endoreduplication and
polyploidization
2) division of cells surrounding the syncitium.
Here again for the same reason as above, inhibition of S phase deems as the
most
efficient way to prevent formation of the feeding site. Other targets are
proteins
involved in GONG 1 transition (re-entry into the cell cycle) G 1 /S transition
and S/G2/G 1
shunting.
Fungal triggering of the formation of new host tissues needed for fungal spore
production may be mediated by the phytohormone cytokinin. Inhibiting fungal
infection and spread of fungal spores can be expected if the disease phenotype
can
be suppressed. This can be achieved by temporarily eliminating the
proliferative
effect of cytokinin on plant cells.
The temporal aspect is obtained by using a promoter inducible by fungal
infection.
The temporal elimination of the cytokinin effect at the G1-stage of the cell
cycle can
be achieved by operably linking to the described promoter of sequences of cell
cycle
genes.
As described above, various cell cycle genes can be used for the construction
of the
chimeric genes and recombinant DNAs of the invention in order to modify the
cell cycle,
e.g., arresting the cell cycle or cell division of a plant cell. For example,
dominant
negative versions of a cell cycle gene can be employed.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
24
The term udominant negative versions", used herein, is defined as a cell cycle
gene as
described above encoding a cell cycle control protein, e.g., a CDK protein
comprising at
least one mutation, e.g., an amino acid substitution, deletion or addition.
Furthermore, in mammals as well as in yeast the function of the WEE1 protein
kinase is antagonistic to CDC25, acting as a mitotic inhibitor by
phosphorylation of
CDC2 on TyrlS (Igarashi, Nature 353 (1991 ), 80-3; Russell and Nurse, Cell 49
(1987), 559-567; Labib and Nurse, Current Biology, 3 (1993), 164-166). A Wee 1
plant homologue from maize, ZmWee1 has recently been identified (Sun, Proc.
Natl.
Acad. Sci. USA 96 (1999), 4180-4185). In fission yeast MIK1 acts cooperatively
with
the WEE1 protein kinase in the inhibitory Tyrl5 phosphorylation of CDC2
(Lundgren,
Cell 64 (1991), 1111-1122). In Xenopus a MYT1 kinase has been identified that
phosphorylates CDC2 at both Tyrl5 and Thr 14 to keep the CDC2 complex in a
mitotic inactive state (Mueller, Science 270 {1998), 86-89).
Thus, another attractive route to obtain pathogen resistant plants according
to the
present invention is by conferring to the giant the capacity to induce and/or
enhance
upon pathogen infection, the expression or activity of at least Wee-kinase,
MIK1 or MYT
or a functional equivalent thereof, thereby increasing the endogenous
phosphorylation
of CDK of the said plant at least the tyrosine at position 15. Wee-kinase is
reviewed in,
e.g., Lew and Kornbluth, supra. This kinase phosphorylates the above-discussed
Y-15
of CDK and may also be responsible for the phosphorylation of the T-14. With
"functional equivalent of Wee-kinase" is meant any endogenous kinase of the
plant
having the function of known Wee-kinase in phosphorylating the respective
tyrosine
residue and optionally the threonine residue of the endogenous plant CDK. The
recently
identified Myt1 kinase (Mueller, Science 270 (1995), p. 86) may therefore be
regarded
as such a functional equivalent. By inducing the expression of the Wee-kinase
upon
pathogen infection, the phosphorylation of CDK will be increased, initiating
the
downregulation of cell division (mitotic activity) and growth, thus obtaining
pathogen
resistance.
Thus, engineering of transgenic plants in accordance with the present
invention
comprises the use of the animal or yeast CDC25, WEE1, MYT1 or MIK1 genes or
more preferably their plant homologues such as Wee1 from maize; see Sun,
supra.
Strategies include overexpressing cell cycle inhibitory genes such as CKI by
use of a
pathogen inducible control sequence described herein and - preferably under
the
control of a pathogen inducible promoter -knockout of cell cycle stimulating
genes
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 such as CDKs by, e.g., RNA antisense or sense constructs, t-DNA insertion,
co-
suppression, dominant negative mutants, homologous recombination technology,
antibody expression etc. described in more detail below.
In the latter strategy the presence, transcription and/or expression of the
chimeric gene
or recombinant DNA molecule of the invention leads to reduction of the
synthesis or the
10 activity of cell cycle proteins or proteins acting on such proteins thereby
resulting in
down modulating the cell cycle and preferably cell division in transgenic
plants
compared to wild type plants.
Therefore, the use of nucleic acid molecules encoding an antisense RNA which
is
i 5 complementary to transcripts of a cell cycle gene, e.g. CDK, in a plant is
also the
subject matter of the present invention. Thereby, complementarity does not
signify that
the encoded RNA has to be 100% complementary. A low degree of complementarity
is
sufficient, as long as it is high enough in order to inhibit the expression of
the target cell
cycle gene upon expression in plant cells. The transcribed RNA is preferably
at least
20 90% and most preferably at least 95% complementary to the transcript of the
cell cycle
gene. In order to cause an antisense-effect during the transcription in plant
cells such
DNA molecules have a length of at least 15 bp, preferably a length of more
than 100 by
and most preferably a length or more than 500 bp, however, usually less than
5000 bp,
preferably shorter than 2500 bp. Standard methods relating to antisense
technology
25 have been described; see, e.g., Klann, Plant Physiol. 112 (1996), 1321-
1330. Also DNA
molecules can be employed which, during expression in plant cells, lead to the
synthesis of an RNA which in the plant cells due to a co-suppression-effect
reduces the
expression of the nucleic acid molecules encoding the described cell cycle
proteins.
The principle of the co-suppression as well as the production of corresponding
DNA
sequences is precisely described, for example, in WO 90/12084. Such DNA
molecules
preferably encode an RNA having a high degree of homology to transcripts of
the cell
cycle genes. It is, however, not absolutely necessary that the coding RNA is
translatable into a protein. The principle of co-suppression effect is known
to the
person skilled in the art and is, for example, described in Jorgensen, Trends
Biotechnol. 8 (1990), 340-344; Niebel, Curr. Top. Microbiol. Immunol. 197
(1995), 91-
103; Flavell, Curr. Top. Microbiol. Immunol. 197 (1995), 43-36; Palaqui and
Vaucheret,
Plant. Mol. Biol. 29 (1995), 149-159; Vaucheret, Mol. Gen. Genet. 248 (1995),
311-317;
de Bome, MoI. Gen. Genet. 243 (1994), 613-621 and in other sources.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
26
Likewise, DNA molecules encoding an RNA molecule with ribozyme activity which
specifically cleaves transcripts of a gene encoding the dephosphorylating
enzyme
can be used. Ribozymes are catalytically active RNA molecules capable of
cleaving
RNA molecules and specific target sequences. By means of recombinant DNA
techniques it is possible to alter the specificity of ribozymes. There are
various
classes of ribozymes. For practical applications aiming at the specific
cleavage of
the transcript of a certain gene, use is preferably made of representatives of
two
different groups of ribozymes. The first group is made up of ribozymes which
belong
to the group I intron ribozyme type. The second group consists of ribozymes
which
as a characteristic structural feature exhibit the so-called "hammerhead"
motif. The
specific recognition of the target RNA molecule may be modified by altering
the
sequences flanking this motif. By base pairing with sequences in the target
molecule
these sequences determine the position at which the catalytic reaction and
therefore
the cleavage of the target molecule takes place. Since the sequence
requirements
for an efficient cleavage are low, it is in principle possible to develop
specific
ribozymes for practically each desired RNA molecule.
In order to produce DNA molecules encoding a ribozyme which specifically
cleaves
transcripts of a cell cycle gene encoding for example a CDK, for example a DNA
sequence encoding a catalytic domain of a ribozyme is bilaterally linked with
DNA
sequences which are homologous to sequences encoding the target protein.
Sequences encoding the catalytic domain may for example be the catalytic
domain of
the satellite DNA of the SCMo virus (Davies, Virology 177 (1990), 216-224 and
Steinecke, EMBO J. 11 (1992), 1525-1530) or that of the satellite DNA of the
TobR
virus (Haseloff and Gerlach, Nature 334 (1988), 585-591). The DNA sequences
flanking
the catalytic domain are preferably derived from the above-described DNA
molecules of
the invention. The expression of ribozymes in order to decrease the activity
in certain
proteins in cells is also known to the person skilled in the art and is, for
example,
described in EP-A1 0 321 201, EP-A1 0 291 533, EP-A2 0 360 257. Selection of
appropriate target sites and corresponding ribozymes as well testing their
activity
can be done as described for example in Steinecke, Ribozymes, Methods in Cell
Biology 50, Galbraith, eds Academic Press, Inc. (1995), 449-460. The
expression of
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
27
ribozymes in plant cells was, for example, also described, in Feyter et al.
(Mol. Gen.
Genet. 250 (1996), 329-338).
Furthermore, the activity of cell cycle genes or their gene products in plant
cells can
be decreased by the so-called "in vivo mutagenesis", for which a hybrid RNA-
DNA
oligonucleotide ("chimeroplast") is introduced into cells by transformation of
cells
TIBTECH 15 (1997), 441-447; WO 95/15972; Kren, Hepatology 25 (1997), 1462-
1468;
Cole-Strauss, Science 273 (1996), 1386-1389). Part of the DNA component of the
RNA-DNA oligonucleotide is homologous to a nucleic acid sequence of an
endogenous cell cycle gene, in comparison to the said nucleic acid sequence it
displays, however, a mutation or contains a heterologous region which is
surrounded
by the homologous regions. By means of base pairing of the homologous regions
of
the RNA-DNA oligonucleotide and of the endogenous nucleic acid molecule
followed
by a homologous recombination the mutation contained in the DNA component of
the RNA-DNA oligonucleotide or the heterologous region can be transferred to
the
genome of a plant cell. This results in a decrease of the activity.
Furthermore, nucleic acid molecules encoding antibodies specifically
recognizing a cell
cycle protein, i.e. specific fragments or epitopes, of such a protein can be
used for
inhibiting the activity of the protein in plants. These antibodies can be
monoclonal
antibodies, polyclonal antibodies or synthetic antibodies as well as fragments
of
antibodies, such as Fab, Fv or scFv fragments etc. Monoclonal antibodies can
be
prepared, for example, by the techniques as originally described in Kohler and
Milstein,
Nature 256 (1975), 495; and Galfre, Meth. Enzymol. 73 {1981), 3, which
comprise the
fusion of mouse myeloma cells to spleen cells derived from immunized mammals.
Furthermore, antibodies or fragments thereof to peptides of the aforementioned
cell
cycle control proteins can be obtained by using methods which are described,
e.g.,
in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring
Harbor, 1988. Expression of antibodies or antibody-like molecules in plants
can be
achieved by methods well known in the art, for example, full-size antibodies
(During,
Plant. Mol. Biol. 15 (1990), 281-293; Hiatt, Nature 342 (1989), 469-470; Voss,
Mol.
Breeding 1 (1995), 39-50), Fab-fragments (De Neve, Transgenic Res. 2 (1993),
227-
237), scFvs (Owen, Bio/Technology 10 (1992), 790-794; Zimmermann, Mol.
Breeding 4
(1998), 369-379; Tavladoraki, Nature 366 (1993), 469-472) and dAbs (Benvenuto,
Plant
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
28
Mol. Biol. 17 (1991 ), 865-874) have been successfully expressed in Tobacco,
Potato
(Schouten, FEES Lett. 415 (1997), 235-241 ) or Arabidopsis, reaching
expression levels
as high as 6.8% of the total protein (Fiedler, Immunotechnology 3 (1997), 205-
216).
In addition, nucleic acid molecules encoding mutant forms of a cell cycle
protein can be
used to interfere with the activity of the wild type protein. Such mutant
forms preferably
have lost their biological activity, e.g., kinase activity and may be derived
from the
corresponding wild-type protein by way of amino acid deletion(s),
substitution(s), and/or
additions in the amino acid sequence of the protein. Mutant forms such
proteins also
encompass hyper-active mutant forms of such proteins which display, e.g., an
increased substrate affinity and/or higher substrate turnover of the same.
Furthermore, such hyper-active forms may be more stable in the cell due to the
incorporation of amino acids that stabilize proteins in the cellular
environment. These
mutant forms may be naturally occurring or genetically engineered mutants, see
also
supra.
The nucleic acid and amino acid sequences for cell cycle proteins can be
arrived, for
example, from the above-described Wee-kinase MIK or MYT proteins. Furthermore,
it is immediately evident to the person skilled in the art that the above-
described
antisense, ribozyme, co-suppression, in vivo mutagenesis, antibody expression
and
dominant mutant effects can also be used for the reduction of the expression
of
genes that encode a regulatory protein such as transcription factors that
control the
expression of cell cycle genes in plant cells. Likewise the described methods
can be
used, for example, to knock-out the activity of regulatory proteins that, for
example,
are necessary for cell cycle genes, e.g., CDKs to become active. Furthermore,
the
above-described methods can be used to knock-out the expression or activity of
the
endogenous wild-type forms of cell cycle genes in plant cells. This would have
the
advantage that a cell cycle mutein in the plant cell does not have to compete
with the
wild-type form and that therefore, lower levels of cell cycle muteins may be
sufficient
so as to achieve the desired phenotype.
The present invention also relates to vectors, particularly plasmids, cosmids,
viruses
and bacteriophages used conventionally in genetic engineering that comprise a
chimeric gene or a recombinant DNA molecule of the invention. Preferably, said
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
z9
vector is a plant expression vector, preferably further comprising a selection
marker
for plants. For example of suitable selector markers, see infra. Methods which
are
well known to those skilled in the art can be used to construct recombinant
vectors;
see, for example, the techniques described in Sambrook, Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel,
Current
Protocols in Molecular Biology, Green Publishing Associates and Wiley
Interscience,
N.Y. (1989), (1994). Alternatively, the chimeric promoters and recombinant
genes of
the invention can be reconstituted into liposomes for delivery to target
cells.
Advantageously, the above-described vectors of the invention comprise a
selectable
and/or scorable marker. Selectable marker genes useful for the selection of
transformed plant cells, callus, plant tissue and plants are well known to
those skilled
in the art and comprise, for example, antimetabolite resistance as the basis
of
selection for dhfr, which confers resistance to methotrexate (Reiss, Plant
Physiol.
{Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the
aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2
(1983), 987-995) and hygro, which confers resistance to hygromycin (Marsh,
Gene
32 (1984), 481-485). Additional selectable genes have been described, namely
trpB,
which allows cells to utilize indole in place of tryptophan; hisD, which
allows cells to
utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85
(1988),
8047); mannose-6-phosphate isomerase which allows cells to utilize mannose
(WO 94/20627) and ODC (ornithine decarboxylase) which confers resistance to
the
ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue, 1987, In: Current Communications in Molecular Biology, Cold
Spring
Harbor Laboratory ed.) or deaminase from Aspergillus terreus which confers
resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995),
2336-
2338).
Useful scorable marker are also known to those skilled in the art and are
commercially available. Advantageously, said marker is a gene encoding
luciferase
(Giacomin, PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121),
green
fluorescent protein (Gerdes, FEES Lett. 389 (1996), 44-47) or f3-glucuronidase
(Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is particularly
useful for
simple and rapid screening of cells, tissues and plants containing a vector of
the
invention.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5
The present invention furthermore relates to host cells comprising a chimeric
gene,
recombinant DNA molecule or a vector according to the invention wherein the
chimeric gene, recombinant DNA molecule or vector is foreign to the host cell.
By "foreign" it is meant that the chimeric gene is either heterologous with
respect to
10 the host cell, this means derived from a cell or organism with a different
genomic
background, or is homologous with respect to the host cell but located in a
different
genomic environment than the naturally occurring counterpart of said gene.
This
means that, if the chimeric gene is homologous with respect to the host cell,
it is not
located in its natural location in the genome of said host cell, in particular
it is
15 surrounded by different genes. The vector or recombinant DNA according to
the
invention which is present in the host cell may either be integrated into the
genome
of the host cell or it may be maintained in some form extrachromosomally. The
host
cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect,
fungal, plant
or animal cells. Preferred cells are plant cells.
In a further preferred embodiment, the present invention provides a method for
the
production of transgenic plants, with a reduced susceptibility to a pathogen
infection
and/or spread thereof comprising the introduction of a chimeric gene,
recombinant
DNA molecule or vector of the invention into the genome of a plant, plant cell
or
plant tissue. For the expression of the cell cycle gene under the control of
the
pathogen inducible promoter in plant cells, further regulatory sequences such
as poly
A tail may be fused, preferably 3' to the heterologous DNA sequence, see also
supra. Further possibilities might be to add Matrix Attachment Sites at the
borders of
the transgene to act as "delimiters" and insulate against methylation spread
from
nearby heterochromatic sequences.
Methods for the introduction of foreign genes into plants are also well known
in the art.
These include, for example, the transformation of plant cells or tissues with
T-DNA
using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of
protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection,
electroporation,
vacuum infiltration, biolistic methods like particle bombardment, pollen-
mediated
transformation, plant RNA virus-mediated transformation, liposome-mediated
transformation, transformation usina wounded nr PnwmP-~iAnrarlArl immat~ pro
embryos, or wounded or enzyme-degraded embryogenic callus and other methods
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
31
known in the art. The vectors used in the method of the invention may contain
further
functional elements, for example "left border"- and "right border"-sequences
of the T-
DNA of Agrobacterium which allow stable integration into the plant genome.
Furthermore, methods and vectors are known to the person skilled in the art
which
permit the generation of marker free transgenic plants, i.e. the selectable or
scorable
marker gene is lost at a certain stage of plant development or plant breeding.
This
can be achieved by, for example cotransformation (Lyznik, Plant Mol. Biol. 13
(1989), 151-161; Peng, Plant Mol. Biol. 27 (1995), 91-104) and/or by using
systems
which utilize enzymes capable of promoting homologous recombination in plants
(see, e.g. WO 97/08331; Bayley, Plant Mol. Biol. 18 (1992), 353-361); Lloyd,
Mol.
Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet. 230 (1991), 170-176;
Onouchi, Nucl. Acids Res. 19 (1991), 6373-6378). Methods for the preparation
of
appropriate vectors are described by, e.g., Sambrook (Molecular Cloning; A
Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, NY).
Suitable strains of Agrobacterium tumefaciens and vectors as well as
transformation
of Agrobacteria and appropriate growth and selection media are well known to
those
skilled in the art and are described in the prior art (GV3101 (pMK90RK),
Koncz, Mol.
Gen. Genet. 204 (1986), 383-396; C58C1 {pGV 3850kan), Deblaere, Nucl. Acid
Res.
13 (1985), 4777; Bevan, Nucleic. Acid Res. 12 (1984), 8711; Koncz, Proc. Natl.
Acad.
Sci. USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol. 20 (1992), 963-976;
Koncz,
Specialized vectors for gene tagging and expression studies. In: Plant
Molecular
Biology Manual Vol. 2, Gelvin and Schilperoort (Eds.), Dordrecht, The
Netherlands:
Kluwer Academic Publ. (1994), 1-22; EP-A-120 516; Hoekema: The Binary Plant
Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V,
Fraley, Crit. Rev. Plant. Sci., 4, 1-46; An, EMBO J. 4 (1985), 277-287).
Although the
use of Agrobacterium tumefaciens is preferred in the method of the invention,
other
Agrobacterium strains, such as Agrobacterium rhizogenes, may be used, for
example if
a phenotype conferred by said strain is desired.
Methods for the transformation using biolistic methods are well known to the
person
skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil,
BiolTechnology 11 (1993), 1553-1558 and Christou (1996) Trends in Plant
Science 1,
423-431. Microinjection can be performed as described in Potrykus and
Spangenberg
(eds.), Gene Transfer To Plants. Springer Verlag, Berlin, NY (1995).
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
32
The transformation of most dicotyledonous plants is possible with the methods
described above. But also for the transformation of monocotyledonous plants
several
successful transformation techniques have been developed. These include the
transformation using biolistic methods as, e.g., described above as well as
protoplast
transformation, electroporation of partially permeabilized cells, introduction
of DNA
using glass fibers, etc.
The resulting transformed plant cell can then be used to regenerate a
transformed
plant in a manner known by a skilled person.
Alternatively, a plant cell can be used and modified such that said plant cell
expresses an endogenous gene capable of modifying the cell cycle under the
control
of the pathogen inducible promoter or vice versa. The introduction of the
pathogen
inducible promoter which does not naturally control the expression of a given
gene or
genomic sequences using, e.g., gene targeting vectors can be done according to
standard methods, see supra and, e.g., Hayashi, Science 258 (1992), 1350-1353;
Fritze and Walden, Gene activation by T-DNA tagging. In Methods in Molecular
biology 44 (Gartland, K.M.A. and Davey, M.R., eds). Totowa: Human Press
(1995),
281-294) or transposon tagging (Chandlee, Physiologia Plantarum 78 (1990), 105-
115).
In general, the plants which can be modified according to the invention can be
derived from any desired plant species. They can be monocotyledonous plants or
dicotyledonous plants, preferably they belong to plant species of interest in
agriculture, wood culture or horticulture interest, such as a crop plant, root
plant, oil
producing plant, wood producing plant, agricultured bioticultured plant, fruit-
producing plant, fodder or forage legume, companion plant, or horticultured
plant,
e.g., such a plant is wheat, barley, maize, rice, carrot, sugar beet, chicory,
cotton,
sunflower, tomato, cassava, grapes, soybean, sugar cane, flax, oilseed rape,
tea,
canola, onion, asparagus, carrot, celery, cabbage, lentil, broccoli,
cauliflower, brussel
sprout, artichoke, okra, squash, kale, collard greens, rye, sorghum, oats,
tobacco,
pepper, grape or potato. Additional species are not excluded.
Thus, the present invention relates also to transgenic plant cells comprising,
preferably stably integrated into the genome, a chimeric gene, a recombinant
DNA
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
33
molecule or vector according to the invention or obtainable by the above-
described
method, wherein the chimeric gene, recombinant DNA a vector is foreign to the
transgenic plant cell. For the meaning of the term "foreign"; see supra.
Furthermore, the present invention also relates to transgenic plants and plant
tissue
comprising the above-described transgenic plant cells or obtainable by the
above-
described method. These plants may show, for example, increased disease
resistance. In a preferred embodiment of the invention, the transgenic plant
upon the
presence of the chimeric gene or the recombinant DNA molecule of the invention
attained resistance or improved resistance against a pathogen the
corresponding
wild-type plant was susceptible to. The term "resistance" covers the range of
protection from a delay to complete inhibition of disease development.
It is also evident from the disclosure of the present invention, that any
combination of
the above-identified strategies can be used for the generation of transgenic
plants,
which due to the presence of a chimeric gene or recombinant DNA molecule of
the
present invention display a novel or enhanced resistance to a pathogen. Such
combinations can be made, e.g., by (co-)transformation of corresponding
nucleic
acid molecules into the plant cell, plant tissue or plant, or may be achieved
by
crossing transgenic plants that have been generated by different embodiments
of the
method of the present invention. Likewise, the plants obtainable by the method
of
the present invention can be crossed with other transgenic plants so as to
achieve a
combination of and another genetically engineered trait.
Any transformed plant obtained according to the invention can be used in a
conventional breeding scheme or in in vitro plant propagation to produce more
transformed plants with the same characteristics and/or can be used to
introduce the
same characteristic in other varieties of the same or related species.
Furthermore, the characteristic of the transgenic plants of the present
invention to
display reduced susceptibility to a plant pathogen can be combined with
various
approaches to confer, e.g., biotic or abiotic stress tolerance.
Thus, due to the findings of the present invention, it is now also possible to
produce
transgenic plants which are less susceptible to pathogens and display further
new
phenotype characteristics compared to naturally occurring wild-type plants,
for
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
34
example, due to the presence of another transgene. Hence, the above-described
chimeric genes and recombinant DNA molecule can be used in combination with
other transgenes that confer another phenotype to the plant. Likewise, it is
possible
to first confer, pathogen resistance to a plant in accordance with the method
of the
invention and to then in an additional step transform such plant in accordance
thereof with a further nucleic acid molecule, the presence of which results in
another
new phenotype characteristic of said plant. Irrespective of the actual
performance of
transformation, the result of the present invention displays at least two new
properties compared to a naturally occurring wild-type plant, that is
increased
resistance to pathogens and; a phenotype that is due to the presence of a
further
nucleic acid molecule in said plants. For example, said phenotype is conferred
by
the (over)expression of homologous or heterologous genes or suppression of
endogenous genes of the plant or their gene products. Some examples for the
(over)expression of homologous or heterologous genes and antisense inhibition
and
co-suppression aiming at manipulating certain metabolic pathways in transgenic
plants are reviews in Herbers (TIBTECH 14 (1996), 198-205). Thus, it is
immediately
evident to the person skilled in the art that the method of the present
invention can
be employed to produce transgenic pathogen resistant plants with any further
desired trait (see for review TIPTEC Plant Product & Crop Biotechnology 13
{1995),
312-397) comprising (i) herbicide tolerance (DE-A-3701623; Stalker, Science
242
(1988), 419), (ii) insect resistance (Vaek, Plant Cell 5 (1987), 159-169),
(iii) virus
resistance (Powell, Science 232 (1986), 738-743; Pappu, World Journal of
Microbiology & Biotechnology 11 (1995), 426-437; Lawson, Phytopathology 86
(1996) 56 suppl.), (vi) ozone resistance (Van Camp, Biotech. 12 (1994), 165-
168),
(v) improving the preserving of fruits (Oeller, Science 254 (1991), 437-439),
(vi)
improvement of starch composition and/or production (Stark, Science 242
(1992),
419; Visser, Mol. Gen. Genet. 225 (1991), 289-296), (vii) altering lipid
composition
(Voelker, Science 257 (1992}, 72-74), (viii) production of (bio)polymers
(Poirer,
Science 256 (1992), 520-523), (ix) alteration of the flower color, e.g., by
manipulating
the anthocyanin and flavonoid biosynthetic pathway (Meyer, Nature 330 (1987},
667-
678, WO 90/12084), (x) resistance to bacteria, insects and fungi (Duering,
Molecular
Breeding 2 (1996}, 297-305; Strittmatter, Bio/Technology 13 (1995), 1085-1089;
Estruch, Nature Biotechnology 15 (1997), 137-141 ), (xi} alteration of
alkaloid and/or
cardiac glycoside composition, (xii) inducing maintaining male and/or female
sterility
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 (EP-A1 0 412 006; EP-A1 0 223 399; WO 93/25695); (xiii) higher longevity of
the
inflorescences/flowers, and (xvi) stress resistance; see, e.g., WO 99/05902
and
WO 97/13843.
Thus, the present invention relates to any plant cell, plant tissue, or plant
which due
10 to genetic engineering displays pathogen resistance obtainable in
accordance with
the method of the present invention and comprising a further nucleic acid
molecule
conferring a novel phenotype to the plant such as one of those described
above.
In yet another aspect the invention also relates to harvestable parts and to
15 propagation material of the transgenic plants according to the invention
which
contain transgenic plant cells described above. Harvestable parts can be in
principle
any useful part of a plant, for example, leaves, stems, fruit, seeds, roots,
flours,
pollen, etc. Propagation material includes, for example, seeds, fruits,
cuttings,
seedlings, tubers, rootstocks, etc.
In addition, the present invention relates to a kit comprising the chimeric
gene, the
recombinant DNA molecule, or the vector of the invention. The kit of the
invention
may contain further ingredients such as selection markers and components for
selective media suitable for the generation of transgenic plant cells, plant
tissue or
plants. Furthermore, the kit may include buffers and substrates for reporter
genes
that may be present in the recombinant DNA or vector of the invention. The kit
of the
invention may advantageously be used for carrying out the method of the
invention
and could be, inter alia, employed in a variety of applications referred to
herein, e.g.,
as research tool. The parts of the kit of the invention can be packaged
individually in
vials or in combination in containers or multicontainer units. Manufacture of
the kit
follows preferably standard procedures which are known to the person skilled
in the
art. The kit or its ingredients according to the invention can be used in
plant cell and
plant tissue cultures. The kit of the invention and its ingredients are
expected to be
very useful in breeding new varieties of, for example, plants which display
improved
properties such as nematode or virus resistance. It is also immediately
evident to the
person skilled in the art that the chimeric gene, recombinant DNA molecule and
vectors of the present invention can be employed to produce transgenic plants
with a
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
36
(further) desired trait (see for review TiPTEC Plant Product & Crop
Biotechnology 13
(1995), 312-397).
An important aspect of the invention is also a method for combating plant
pathogens
which comprises expressing a cell cycle gene in a plant under the control of a
plant
pathogen inducible control sequence such as a promoter region. Preferred
strategies
in combating with different pathogens are as follows.
Nematodes
A preferred embodiment of the invention with respect to nematodes includes a
pathogen inducible promoter operably linked to a dominant negative mutant of
cdc2a
or CDC7.
A further preferred embodiment of the invention with respect to nematodes
includes
a pathogen inducible promoter operably linked to Rb.
A still further preferred embodiment of the invention with respect to
nematodes
includes a pathogen inducible promoter operably linked to a dominant negative
E2F
that has a mutated activation domain, to an antisense E2F or to genes encoding
components of the SCF complex (e.g. Skp, cullin, F box protein) involved in
E2F
proteolysis.
In another preferred embodiment with respect to nematodes the present
invention
relates to a pathogen inducible promoter operably linked to cyclin D with a
mutated
RB binding domain.
A still further preferred embodiment of the invention with respect to
nematodes
includes a pathogen inducible promoter operably linked to a CKI or CKS.
Geminivirus
A preferred embodiment of the invention with respect to gemini viruses
includes a
pathogen inducible promoter operably linked to a Rb, to a dominant negative
E2F
which has a mutated activation domain, to an antisense E2F, to genes encoding
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
37
components of the SCF complex (e.g. Skp, cullin, F box protein) involved in
E2F
proteolysis, to antisense DNA polymerase, or to an antisense PCNA.
Fungi
A preferred embodiment of the invention with respect to fungi includes a
pathogen
inducible promoter operably linked to an antisense cyclin D, to a dominant-
negative
CDK mutant protein acting at G1, to Rb, to cyclin B, or to Wee 1 kinase.
It is to be understood that the skilled person, aware of the above teaching,
will be able
to apply numerous techniques to confer to a plant the capacity to downregulate
cell
cycle progress as is discussed above upon pathogen infection. Thus, the
present
invention generally relates to the use of the above described cell cycle genes
and in
particular chimeric genes, recombinant DNAs and vectors of the invention for
conferring pathogen resistance to a plant. Furthermore, the present invention
relates
to the use of a pathogen inducible promoter for the expression a cell cycle
gene and
to the use of a cell cycle gene or a pathogen inducible promoter for the
construction
of a chimeric gene, recombinant DNA molecule, vector of the invention or for
the
generation of a host cell or plant cell of the invention.
These and other embodiments are disclosed and encompassed by the description
and examples of the present invention. Further literature concerning any one
of the
methods, uses and compounds to be employed in accordance with the present
invention may be retrieved from public libraries, using for example electronic
devices. For example the public database "Medline" may be utilized which is
available on the Internet, for example under
http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and
addresses, such as http://www.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/,
http://www.fmi.ch/biology/research tools.html, http://www.tigr.org/, are known
to the
person skilled in the art and can also be obtained using, e.g.,
http://www.lycos.com.
An overview of patent information in biotechnology and a survey of relevant
sources
of patent information useful for retrospective searching and for current
awareness is
given in Berks, TIBTECH 12 (1994), 352-364.
CA 02330550 2000-12-14
WO 99/bb055 PCT/EP99/04139
38
In order to clarify what is meant in this description by some terms a further
explanation
is hereunder given.
Reference herein to a "promoter" is to be taken in its broadest context and
includes
the transcriptional regulatory sequences including the TATA box which is
required for
accurate transcription initiation, with or without a CCAAT box sequence and
additional regulatory elements (i.e. upstream activating sequences, enhancers
and
silencers) which alter gene expression in response to developmental and/or
external
stimuli, or in a tissue-specific manner. The term "promoter" also includes the
transcriptionai regulatory sequences of a classical eukaryotic genomic gene, a
classical prokaryotic gene, (in which case it may include a -35 box sequence
and/or
a -10 box transcriptional regulatory sequences) or viral genes. The term
"promoter" is
also used to describe a synthetic or fusion molecule, or derivative which
confers,
activates or enhances expression of a nucleic acid molecule in a cell, tissue
or
organ. Promoters may contain additional copies of one or more specific
regulatory
elements, to further enhance expression and/or to alter the spatial expression
and/or
temporal expression of a nucleic acid molecule to which it is operably
connected. For
example, copper-responsive, glucocorticoid-responsive, dexamethasone-
responsive
or tetracycline-responsive regulatory elements may be placed adjacent to a
heterologous promoter sequence driving expression of a nucleic acid molecule
to
confer copper inducible, glucocorticoid-inducible, dexamethasone-inducible, or
tetracycline-inducible expression respectively, on said nucleic acid molecule.
The terms "DNA molecule", "polynucleotide", "DNA sequence", "nucleic acid
sequence" or "nucleotide sequence" are interchangeable and as used herein
refer to
a polymeric form of nucleotides of any length unless otherwise specified. This
term
refers only to the primary structure of the molecule. Thus, this term includes
double-
and single-stranded DNA. It also includes known types of modifications, for
example,
methylation, "capsH substitution of one or more of the naturally occurring
nucleotides
with analogs.
With "recombinant DNA molecule" or "chimeric gene" is meant a hybrid DNA
produced by joining pieces of DNA from different sources.
CA 02330550 2000-12-14
WO 99/66055 PCTIEP99/04139
39
With "pathogen" is meant those organisms that have a negative effect on the
physiological state of the plant or a part thereof. Some pathogens are for
instance
nematodes, viruses, bacteria, fungi, insects and parasitic plants.
"Plant cell cycle genes" are cell cycle genes originally present or isolated
from a
plant or a part thereof.
"Cyclin-dependent protein kinase complex" means the complex formed when a,
preferably functional, cyciin associates with a, preferably, functional cyclin
dependent
kinase. Such complexes may be active in phosphorylating proteins and may or
may
not contain additional protein species.
"Cell-cycle kinase inhibitor or cyclin dependent kinase inhibitor" (CKI) is a
protein
which inhibits CDK/cyclin activity and is produced and/or activated when
further cell
division has to be temporarily or continuously prevented.
"Plant cell" comprises any cell derived from any plant and existing in culture
as a
single cell, a group of cells or a callus. A plant cell may also be any cell
in a
developing or mature plant in culture or growing in nature.
"Plants" comprises all plants, including monocotyledonous and dicotyledonous
plants.
"Expression" means the production of a protein or nucleotide sequence in the
cell
itself or in a cell-free system. It includes transcription into an RNA
product, post-
transcriptional modification and/or translation to a protein product or
polypeptide from
a DNA encoding that product, as well as possible post-translational
modifications.
~Operably linked" refers to a juxtaposition wherein the components so
described are
in a relationship permitting them to function in their intended manner. A
control
sequence "operably linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions compatible with
the
control sequences. In case the control sequence is a promoter, it is obvious
for a
skilled person that double-stranded nucleic acid is preferably used.
"Control sequence" refers to regulatory DNA sequences which are necessary to
affect the expression of coding sequences to which they are ligated. The
nature of
such control sequences differs depending upon the host organism. In
prokaryotes,
control sequences generally include promoters, ribosomal binding sites, and
terminators. In eukaryotes generally control sequences include promoters,
terminators and enhancers or silencers. The term "control sequence" is
intended to
include, at a minimum, all components the presence of which are necessary for
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 expression, and may also include additional advantageous components and
which
determines when, how much and where a specific gene is expressed.
The terms "protein" and "polypeptide" used in this application are also
interchangeable. "Polypeptide" refers to a polymer of amino acids (amino acid
sequence) and does not refer to a specific length of the molecule unless
otherwise
10 specified. Thus, peptides and oligopeptides are included within the
definition of
polypeptide. This term does also refer to or include post-translational
modifications
of the polypeptide, for example, glycosylations, acetylations,
phosphorylations and
the like. Included within the definition are, for example, polypeptides
containing one
or more analogs of an amino acid (including, for example, unnatural amino
acids,
15 etc.), polypeptides with substituted linkages, as well as other
modifications known in
the art, both naturally occurring and non-naturally occurring.
"Transformation" as used herein, refers to the transfer of an exogenous
polynucleotide into a host cell, irrespective of the method used for the
transfer. The
polynucleotide may be transiently or stably introduced into the host cell and
may be
20 maintained non-integrated, for example, as a plasmid, or alternatively, may
be
integrated into the host genome. Many types of vectors such as recombinant DNA
molecules or chimeric genes according to the invention can be used to
transform a
plant cell and many methods to transform plants are available. Examples are
direct
gene transfer, pollen-mediated transformation, plant RNA virus-mediated
25 transformation, Agrobacterium-mediated transformation, liposome-mediated
transformation, transformation using wounded or enzyme-degraded immature
embryos, or wounded or enzyme-degraded embryogenic callus. All these methods
and several more are known to persons skilled in the art. The resulting
transformed
plant cell can then be used to regenerate a transformed plant in a manner
known by
30 a skilled person.
Two genes are "functional homologous" when the respective encoded proteins
can,
at least in part, be interchanged in an in vitro and/or in vivo assay
concerning
function.
"Sense strand" refers to the strand of a double-stranded DNA molecule that is
35 homologous to a mRNA transcript thereof. The "anti-sense strand" contains
an
inverted sequence which is complementary to that of the "sense strand".
"Dominant negative version or variant" refers to a mutant protein which
interferes
with the activity of the corresponding wild-type protein.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
41
A more detailed description of the invention, for the sake of clarity, is
disclosed
hereinafter.
The Fi4ures show:
Figure 1: A map of the mutations introduced into both A. thaliana CDKs is
presented
in Figure 1 and characterized as mutant alleles of CDC2aAt and
CDC2bAt. The bar in the middle represents the complete coding region.
The amino acid sequences of the mutated regions (in one-letter code) are
given below and above the bar for the plant CDKs and fission yeast
CDC2, respectively. The dots above the sequence indicate the mutated
amino acids with the arrows pointing to the corresponding changes.
Figure 2: Figure 2 shows the ARM1-alll plasmid containing a 3.7 kb ARM1
promoter fragment in pBluescript.
Figure 3: Figure 3 gives a schematic representation of the results obtained in
the
initial screening of 18 transformed potato lines carrying the P0728-
cdc2and construct: the x-axis shows the line number whereas the y-axis
represents the ratio of galls to inoculated root tips. WT-D = wild type
Bintje: S = Solanum.
The present invention is further described by reference to the following non-
limiting
figures and examples.
Unless stated otherwise in the Examples, all recombinant DNA techniques are
performed according to protocols as described in Sambrook et al. (1989),
Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY or in
Volumes 1 and 2 of Ausubel et al., (1994), Current Protocols in Molecular
Biology,
Current Protocols. Standard materials and methods for plant molecular work are
described in Plant Molecular Biology Labfase (1993) by R.D.D. Croy, jointly
published by BIOS Scientific Publications Ltd. (UK) and Blackwell Scientific
Publications (UK).
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
42
EXAMPLES - NEMATODES
General Methods
A. Nematode cultures and hatching procedures
Root knot nematode (Meloidogyne incognita) cultures were maintained in vitro
on
tomato (Lycopersicon esculentum) hairy roots continuously subcultured on
hormone-
free Gamborg's B5 medium (Flow Laboratories, Bioggio, Switzerland; pH 6.2)
supplemented with 2% sucrose and 1.5% Bacto agar (Difco, Detroit, Ml).
Hatching was stimulated by putting galls (M. incognita) on 70 tun nylon sieves
(Falcon 2350 Cell Strainer; Becton Dickinson, Bedford, MA) submerged in
sterile de
ionized water.
Stocks of H. schachtii are maintained on the host plant Sinapsis alba on Knop
medium (Sijmons et al., 1991 ). Hatching is stimulated by putting cysts on 70
t.im
nylon sieves (Flacon 2350 Cell Strainer) submerged in filter sterile root
exudate
extracted from rapeseen (Brassica napus).
Potato cyst nematodes (Globodera pallida and G. rostochiensis) are propagated
in
soil for the purpose of building up a sufficient stock to conduct resistance
tests on
production crops. Potato tubers are planted in 1 L pots (2 tubers/pot) filled
with a soil
sand mixture (2:1 ratio) to which a slow-release fertilizer is added. Each pot
is
inoculated with an average of 100 cysts (an expected 20,000 infective J2-
nematodes) at the time of planting. Pots were placed in trays (containment)
lined
with a layer of absorptive material and watered via this layer only. Plants
are grown
in a growth chamber (19°C day, 14°C night, 60% humidity and
16h/8h day-night
regime). After 10 weeks cysts can be harvested from the pots by rinsing the
soil with
water and collect the floating cysts.
B. In vitro inoculations of A. thaliana with cyst and root knot nematodes
S2 seeds can be sown directly on selective Knop medium (Sijmons et al., 1991
). On
the other hand, S, plants frequently showed abnormal growth when cultured for
2 weeks or longer on Knop medium, impeding sound analysis of inoculation and
staining results after this time.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
43
Surface-sterilized seeds (2 min in 70% EtOH and 15 min in 5% sodium
hypochlorite)
were germinated on germination medium (Valvekens et al., 1988) supplemented
with
either 50 mg L-' kanamycin monosulfate (Sigma) or 20 mg L-' hygromycin B
(Calbiochem, La Jolla, CA). Two-week-old seedlings were subsequently
transferred
to and lined up on a thin layer of Knop medium. Petri dishes were placed
slightly
tilted to promote unidirectional root growth. After 2 more days of growth at
22°C (16
hr-light/8-hr-dark cycle), roots were inoculated with 5- to 7-day-old hatched
beet cyst
or root knot nematode second-stage juveniles at an average density of 20
juveniles
per root system. The plants were then incubated again under the same tissue
culture
conditions. Five to ten plants were examined for the presence of GUS activity
4 to 6
days post-inoculation (dpi).
In vitro inoculation of potato with root knot nematodes: Top and internode
segments
from 3 week old potato transgenic lines. Per line, 5 to 8 segments are lined
up in a
petridish (Falcon 1013, Becton Dickinison) on 40 ml Knop medium. Two weeks
later
roots are grown long enough and are inoculated with 10 second stage root-knot
juveniles (Mi) per root tip.
In vitro inoculation of tomato hairy roots with root-knot nematodes: Tomato
hairy
roots are subcultured every 3-4 weeks in small petridishes (Falcon 1005) on 50
ml
Gamborg's B5 medium. Two to three weeks after root transfer to fresh B5,
approximately 10 root tips are inoculated with 10 J2 root knot juveniles.
C. Nematode inoculation of soil-grown plants
For cyst and root knot nematode soil inoculations, 2-week-old Arabidopsis
seedlings
were transferred to a 1:2 mixture of cutting soil (M. Snebbout s.a., Kaprijke,
Belgium)
and potting soil (M. Snebbout s.a.) in open translucent plastic tubes. By
placing
these tubes slanting in rectangular flower boxes, the roots were forced to
grow along
one side of the tube, allowing more controlled inoculations and reproducible
infections. Inoculations were performed after 2 more weeks of growth at
22°C and
16 hr of light by injecting a suspension containing 250 second-stage juveniles
(5 to 7
days after hatching) of beet cyst or root knot nematodes in 1.5 ml H20 per
root
system.
CA 02330550 2000-12-14
WO 99/66055 PCTlEP99/04139
44
D. GUS histochemical assay
Histochemical localization of GUS activity was performed using the substrate 5-
bromo-4-chloro-3-indolyl glucuronide (X-gluc: Europa research products, Ely,
U.K.)
according to Jefferson (1987) with minor modifications: 50 ~.~L of X-gluc (20
mg in
1 mL of N,N dimethylformamide) was diluted to a final concentration of 2 mM in
1 mL of 0.1 M NaP04, pH 7.2. Oxidative dimerization of the produced indoxyl
derivative was enhanced by adding the oxidation catalyst K+
ferricyanide/ferrocyanide to a final concentration of 0.5 mM. Incubation of
whole
plantlets in phosphate buffer was preceded by a short treatment (15 to 30 min)
with
90% ice-cold acetone followed by several washes with 0.1 M sodium phosphate,
pH
7.2. The GUS reaction was incubated overnight at 37° C. Stained tissues
were
subsequently fixed for a few hours to overnight in 2.5% glutaraldehyde (Agar
Scientific Ltd., Stansted, U.K.) at 4° C to prevent diffusion of the
GUS product during
the subsequent incubation in chlorallactophenol (2:1:1 mixture of chloral
hydrate,
lactic acid, and phenol) (Beeckman and Engler, 1994). Incubation of the
material in
chlorallactophenol removes all pigments and brown phenolics producing
transparent
tissues which were further monitored for GUS activity using a dissecting light
microscope (Jenalumar; Zeiss, Oberkochen, Germany).
E. Stable transformation with A. tumefaciens
Two potato varieties Bintje and Desiree can be transformed using A.
tumefaciens
carrying the required promoter+cell cycle gene construct. Two protocols by (a)
Kumar (1991) with some modifications as communicated by Steve Miilam (SCRI)
and (b) De Block (1988) are suitable to perform Desiree and Bintje
transformation.
These protocols are applied to leaf material and petiole explants
respectively.
F. Transformation of tomato
Methods of transformation are known to the person skilled in the art. An
example
includes the relatively short transformation procedure using Agrobacterium
rhizogenes (Karimi et al.). Hereto, tomato leaf explants are incubated with a
solution
A rhizogenes carrying the required promoter+cell cycle gene construct.
Developing
hairy roots on selective medium are subcultured and maintained on Gamborg's B5
medium. Other methods of tomato transformation using Agrobacterium tumefaciens
include that of McCormick et al., 1986.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5
EXAMPLE 1: Nematode resistance in Arabidopsis
1.1 CDC2aAt.DN mutant
A mutation, referred to as DN, corresponding to a dominant negative mutant of
the
10 S. pombe CDC2 (Labib 1995 a, b) was introduced in A. thaliana CDC2aAt cDNA -
the resultant mutant form called CDC2aAt.DN (substitution of Asn146 for
Asp146) (see
Figure 1 ). The mutants were obtained by site-directed mutagenesis as follows.
CDC2aAt and CDC2bAt cDNAs were cloned in pGem7Z-f- (Promega, Madison, WI)
and in pUCl8, respectively. The site-directed mutagenesis was performed with
the
15 use of the ExSite PCR-based site-directed mutagenesis kit (Stratage~e, La
Jolla,
CA) according to the manufacturer's instructions. The primers used to
introduce the
mutations were (the mutagenised residues being underlined):
5'-AATTTGGGTCTTGGTCGT-3' (SEQ ID NO: 1 ) and 5'-AGCAATCTTAAGAAGCT-
CTT-3' (SEQ ID NO: 2) for CDC2bAt.DN. The fidelity of the mutagenesis was
20 confirmed by sequencing.
1.2 Construction of expression cassettes
The mutant cDNAs fused to the NOS polyadenylation site were ligated as
Ncol/Mlul
blunt-end fragments to the EcoRl digested (filled-in) plasmid pArm1-alll to
produce
25 transcriptional fusions of the Arm1 promoter and the mutant cDNA. The
resulting
expression cassettes composed of the Arm1 promoter, mutant CDC2aAt cDNAs and
NOS polyadenylation site are generally named Arm1-cdcmutand were transferred
as
Xbal fragments into the Xbal site of the binary vector pGSC1704.
ARM1 or Att0001 is an A. thaliana line containing an in vivo ~i-glucuronidase
fusion
30 that is highly activated in early stages of nematode infection sites
(Barthels et al.,
1997). This article also describes the isolation and confirmation of the
promoter that
is responsible for this expression pattern. The ARM1-alll plasmid contains a
3.7kb
ARM1 promoter fragment in pBluescript (see Figure 2).
35 1.3 Arabidopsis transformation and expression analysis
The binary constructs were introduced in A. tumefaciens C58C1 RifR(pGV2260) by
electroporation with subsequent selection for streptomycin/spectinomycin
resistance.
To confirm the presence and integrity of the plasmids the DNA extracted from
the
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
46
streptomycin/spectinomycin resistance clones was retransformed in E. Coli X~1-
Blue
and the plasmid DNA from the resultant clones was subjected to restriction
digest
analysis with BamHl/Kpnl. A. thaliana Col-O plants were transformed using the
inflorescence infiltration method. Transgenic plants were selected after seed
germination in the presence of hygromycin.
To verify the functionality of the expression cassettes, RNA was prepared from
the
transgenic plants upon induction of the Arm 1 promoter with auxin and
subjected to
RNA blot analysis. Of the 12 lines tested 10 possessed detectable expression,
but
only in three lines the expression level of the transgene was comparable with
the
level of the endogenous CDC2aAt transcript.
1.4 Nematode infection assays
Six weeks after infection, the plants were scored visually for the number of
successful infections and compared to control plants. If the infections are
done in
vitro, scoring can already be done at a much earlier stage (e.g. two weeks
after
inoculation). Plant lines are considered resistant when they show a
significantly
decreased susceptibility to plant pathogenic nematodes; i.e. a significant
decrease in
the number of females or cysts found on roots of the transgenic plants versus
the
number of females or cysts found on the roots of control plants and/or a
significantly
reduced number of nematode feeding sites (for example galls) and/or a
significant
reduction in egg production and/or a significant reduction of viable nematodes
in
these eggs. Susceptible/resistance classification according to the number of
maturing females is standard practice for both cyst- and root- knot nematodes
(e.g.
LaMondia, 1991 ).
1.5 Results - reduction of nematode infection.
The results of nematode infection assays in vitro are summarized in Tables 3
and 4.
In general a correlation could be seen between the expression level of the
transgene
and the effect on nematode infection. For example in DN-1 no transgene
expression
could be detected and the number of galls/cysts and viable progeny is similar
to the
control, while DN-2 with a higher expression and especially DN-3 have a
reduced
level of nematode reproduction. This reduction does not only hold true for the
number of cysts/egg masses per plant but also for the number of infective
juveniles
hatching from the eggs in those cysts/egg masses. For example DN-3 produced an
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
47
M. incognita progeny of on average 209 juveniles from one plant, DN-2 on
average
745 while the control produced on average 1729 juveniles per plant.
When the experiments were done in soil, a significant decrease in production
of
eggs was also seen after inoculation of transgenic plants in comparison with
the
control plants.
EXAMPLE 2: Nematode resistance tests in potato
To engineer nematode resistance a construct was made based on the A. thaliana
0728 promoter in combination with the A. thaliana cdc2aDN gene. Potato
transgenic
lines (Desiree and Bintje) have been made harboring a Po~28-cdc2aDN construct
(pTHW728-cdc2a-DN; for the transformation method: see General Methods). 522
Desiree plants giving +/- 1125 petiole explants and 423 Bintje plants giving
+/- 1200
petiole explants and a comparable amount of leaf material was using in two
transformation procedures (see General Methods). Approximately 260 Desiree and
85 Bintje transgenic fines have been generated.
In an initial screen, 18 independent putative transformed lines have been
tested for
nematode resistance. Therefore 6 to 9 clones (internode segments and tops) of
each
of the 18 selected lines were grown individually on petridishes (145/20mm;
greiner,
Frickenhausen, Germany) containing 40m1 solid Knop medium. Similarly, 11 wild
type Desiree and 10 wild type Bintje tops and internode segments were grown.
Two
weeks after placing the potato segments on knop, emerged root tips were
inoculated
with 10 J2 M. incognita per tip. Reproducible amounts of inocula were obtained
by
mixing juvenile populations with 0.3% low melting agarose (Gibco-BRL, Grand
Island, N.Y.) in de-ionized water. Three days after nematode inoculation,
plants were
scored for gall formation (see Table 5 and Figure 3)
From Table 5 we can remark that lines S5-1 and S11-13 show a remarkably lower
infection rate.
These lines can be further tested with a higher number of clones (replicas).
Similarly
the lines can be inoculated with the potato cyst nematode Globodera pallida or
Globodera rostochiensis (see General Methods).
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
48
EXAMPLE 3: Nematode resistance
Transformation of promoter-cell cycle constructs in tomato
Constructs consisting of a CKI and a pathogen inducible promoter (nematode)
(such
as pAtt0728, pAtt1712), are transformed into tomato (see General Methods).
About
100 transformants per construct are generated and analyzed by PCR for
transgene
integration. 50 transformants/construct are selected for seed production by
self
pollination. The F~ progeny are analyzed for stability of transgene expression
(by RT
PCR) and for the number of transgene integration sites (by segregation
analysis of
the selectable marker). Based on these results, 20 lines/construct (10
plants/line) are
selected for F2 production. Amongst the F2 populations, homozygous lines are
selected for nematode resistance tests.
Nematode resistance tests
Nematode tests are performed on homozygous progeny of 20 independent
transformants/constructs and on the same amount of control plants (e.g.
transformed with empty vector). Tests are done in vitro and in soil. In vitro
plants are
grown for several weeks until the roots are sufficiently developed. After
infection with
Meloidogyne incognito and Heterodera schachtii, plants are cultivated further
in a
sterile growth room and regularly inspected for development of eggmasses or
cysts.
One to two months after infection, eggmasses and cysts are counted and data
are
analyzed statistically. For analysis in soil, plantiets that were germinated
in vitro are
transferred to pots with soil and infected with approx. thousand nematodes.
After 2
months, plants are harvested for analysis of eggmasses or cysts.
In addition to these data on nematode reproduction, microscopical studies are
undertaken in order to follow the infection process, the development of
feeding sites,
and the production of fertile nematodes. Microscopical analysis includes i)
counting
number of feeding sites and rating their size; ii) staining of nematodes (e.g.
with acid
fuchsin) to determine the stage of nematode development; iii) staining of
eggmasses
(e.g. with Phloxine B) to rate egg production.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
49
Phenotyaic analysis of trans4enic plants
Transgenic lines that show improved resistance against nematodes are analyzed
in
more detail for phenotypes under normal growth conditions. Evaluation of
effects of
transgene expression on plant structure and productivity: analysis of growth
rate,
total biomass production, root versus leaf biomass, number and size of root,
branching of roots and stem, leaf shape, microscopical analysis of tissue
anatomy.
EXAMPLE 4: Resistance to geminivirus
4.1 Construction of genetic construct
Constructs containing modified geminiviral CP promoters and an antisense
sequence to the rice PCNA are transformed in Licopersicum escuiemtum by
procedures well known to those skilled in the art.
The pathogen inducible promoter consists of two CLE elements coupled to the
minimum 35S promoter from CaMV as described in Ruiz-Medrano et al. 1999. A
further promoter regulatory element relevant in phloem cell, the silencer-like
element, is introduced either upstream the CLE elements or downstream the PCNA
sequence. The silencer-like elements can be obtained for example from TGMV
(Sunter and Bisaro, 1997) or from other Geminiviruses like PHV (Torres-
Pacheco,
1993) by PCR amplification of the corresponding DNA fragment of approximately
300 by with appropriate reverse and complementary primers like:
Sil-1: 5'-cccaagcttctccactagccgtattttg-3' (SEQ ID NO: 3)
Sil-2: 5'-gcgcgtcgacttcctataaagactacctca-3' (SEQ ID NO: 4)
It is expected that upon geminiviral infection the promoter is upregulated in
mesophyll tissue due to the presence of CLE elements and also in phloem cell
due
to the silencer-like geminiviral responsive element.
The sequence of the PCNA gene is very conserved so sequences of different
origin
can be used as for example the PCNA gene from Rice (Oriza sativa) EMBL no.
X54046 which can be obtained by RT-PCR performed on cDNA from rice cell
suspensions using appropriate downstream and upstream primers. Cell and
molecular biology techniques involved are well known to those skilled in the
art.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 4.2 Transformation of tomato
About 50 independent transformants are generated. The F1 progeny is
characterized
respect to transgene integration site (by segregation analysis of the
selectable
marker), transgene copy number (by Southern blots) and transgene expression by
Northern blots. Based on the results a minimum of 20 lines self-pollinated to
obtain
10 F2 production.
4.3 Geminivirus inoculation
Sensitivity to geminiviral infection is carried out in primary regenerants, F1
and F2
progeny. Resistance is analyzed in whole plants and also in leaf discs
explants to
15 assay interference with replication. As most geminiviruses are not
transmitted
mechanically the infection is carried out by agroinoculation technique
(Grimsley et
al., 1986). As inocula Agrobacterium solution carrying multimers of
geminiviral
clones is used. An example of the clones and bacterial solution used for TYLCV
is
described in Kheyr-Pour et al., (1991).
Plants of three to four weeks old are inoculated with the Agrobacterium
solution with
the aid of a 1 ml syringe in the petioles of the three younger leafs. Plants
are
transferred to 24° C, 16 h light and 70% humidity. 25 plants/line are
used in each
experiment.
4.4 Resistance assessment
Progression of the disease is followed recording symptom development. Three
weeks post inoculation total DNA is extracted from young leaves and analyzed
on
southern blots using a viral specific probe to detect total amount and type of
viral
DNA forms accumulated in different lines. Percentage of plants infected,
symptoms
development and viral accumulation is recorded for each line.
Viral replication capacity in each line is determined -from the leaf disc
assays. An
Agrobacterium containing the viral constructs is grown for 48 h at 27°
C. The
bacterial solution is washed and resuspended in MS media. The tomato leaves
are
sterile cut into leaf discs of approximately 1 cm and mixed with the
agrobacterium
solution for a short period. Leaf discs are transferred to appropriate culture
media
and samples are taken at 5 and 7 dpi.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
51
Total DNA is extracted from the leaf discs and viral DNA forms accumulation is
analyzed by Southern blot as previously described.
Initially resistance to TYLCV, an important tomato pathogen will be assayed.
Based on the results, plants from resistant lines will be challenged with
other
geminiviruses, closely and distantly related to TYLCV.
Selected resistant plants will be further analyzed or phenotypic
characteristics, like
plant life cycle, growth rate, plant architecture, fruit/seed production
before and after
viral inoculation and also under different environmental conditions.
EXAMPLE 5: Strategies to inhibit fungal infection using the cell cycle
5.1 Promoter-cell cycle construct and plant transformation in Arabidopsis
thaliana
A suitable promoter inducible in roots by fungal infection can be the promoter
of the
tobacco EAS4 gene encoding a sesquiterpene cyclase (Yin et al., 1997). A
dominant
negative version of the Cdc2a kinase (Hemerly et al., 1995) can be used as the
cell
cycle gene. The gene construct is introduced into a binary vector such as pain
and
this vector transformed into Agrobacterium tumefaciens. The floral dip method
is
used for transformation of Arabidopsis thaliana C24 (Clough and Bent, 1998).
5.2 Fundai resistance test
Resistance to Plasmodiophora brassicae of selected Arabidopsis lines
transformed
with the construct described above and of untransformed control lines can be
assessed as described by Holtorf et al., (1998): A field isolate
Plasmodiophora
brassiscae is used to infect 10 days old A thaliana C24 seedlings grown in the
greenhouse. They are then inoculated with 0.5 ml of a P. brassicae spore
suspension in 50 mM potassium phosphate buffer (pH 5.5) containing 10'
spores/ml.
After infection, plants are further cultivated in the greenhouse. Roots are
harvested
at 2, 3, 4, 5 and 6 weeks after inoculation. About 50 plants of each line are
used per
time point in one experiment. Three independent experiments are performed. For
each time point the infection rat is calculated as the proportion of plants
which
showed macroscopically detectable root hypertrophy.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
52
5.3 Phenotypic analysis of transaenic plants
Although in tobacco the EAS4 promoter is apparently not active in any tissue
under
normal growth conditions (Yin et al., 1997). Arabidopsis transgenic plants
with the
EAS4 promoter-dominant negative Cdc2 are preferably analyzed in detail for
deviating phenotypes. This is essentially done as described in example 3.
It will be clear that the invention may be practiced otherwise than as
particularly
described in the foregoing description and examples. Numerous modifications
and
variations of the present invention are possible in light of the above
teachings and,
therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent
applications,
journal articles, abstracts, laboratory manuals, books, or other disclosures)
in the
Background of the Invention, Detailed Description, and Examples is hereby
incorporated by reference.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
53
Table 1 - Examples of nematodes affecting the cell cycle:
EffectNematode Host Reference
Hypertrophy
of
nuclei
and
DNA
synthesis
Pratylenchus penefransBroad bean rootsVovlas and Troccoli 1990
Trophotylenchus floridesisPinus Clausa Cohn and Kaplan 1983
roots
Gracilacus hamicaudafaParenchyma tissueCid del Prado and Maggenti
of 1988
the vascular
cylinder
of redwood tree
roots
Globodera rostochiensisSolanum tuberosumJones and Northcote,
(potato) 1972
Hererodera giycines Glycine max (soya)Endo, 1971; Jones and
Dropkin,
1975
Rotylenchulus spp Glycine max (soya)Jones and Dropkin, 1975;
Jones,
1981
Nacobbus spp Jones, 1981
Meloidodera floridensis Jones, 1981
Tyienchulus semipenetrans Jones, 1981
Multinucleate
cells
by
repeated
mitosis
without
cell
division
Xiphinema index Ficus carica Wyss et al. 1980
roots
Xiphinema divericaudafumRoot tip galls Griffiths and Robertson
of 1988
strawberry and
ryegrass
Acytokinetic
mitoses
and
hypertrophy
of
the
nuclei,
DNA
synthesis
Meloidogyne javanical_ycopersicon Bird, 1961; 1962
Meloidogyne incognitaesculentum (tomato)Starr, 1993
M. incognita Lactuca sativa Starr, 1993
{lettuce)
M. incognita Gossypium hirsutumRohde and McClure, 1975
(cotton)
M. javanica Vicia faba (broadHuang and Maggenti, 1969
bean) Bird, 1973
M. incognita Vicia faba (bean)Starr, 1993
M. incognita & javanicaImpatiens balsaminaJones and Payne, 1978
M. incognita Glycine max (soya)Jones and Dropkin, 1975
M. incognita Pisum sativum Starr, 1993
(pea)
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
54
Table 2 - Pathogen inducible promoters
Name Pathogen Reference
RB7 Root-knot nematodes US5760386 - North Carolina State
{Meloidogyne spp.) University; Opperman et al.,
1994.
PR-1, 2, fungal, viral, bacterialWard et al., 1991; Reiss, 1996;
3, Lebel et
4, 5, 8, al., 1998; Melchers et al., 1994;
11 Lawton
et al., 1992
HMG2 nematodes WO 95/03690 - Virginia Tech Intellectual
Properties Inc .
Abi3 Cyst nematodes (Heteroderaunpublished
spp~)
ARM1 nematodes Barthels et al., 1997
WO 98/31822 - Plant Genetic Systems
Att0728 nematodes Barthels et al., 1997
PCT/E P98/07761
Att1712 nematodes Barthels et al., 1997
PCT/EP98/07761
Gst1 Different types of pathogensStrittmatter et al., 1996.
LEMMI nematodes WO 92121757 - Plant Genetic Systems
CLE geminivirus PCT/EP99/03445 - CINESTAV
PDF1.2 Fungal including AlternariaManners et al., 1998
brassicicola and Botryfis
cinerea
Thi2.1 Fungal - Fusarium Vignutelli et al., 1998
oxysporum f sp. matthiolae
DB#226 nematodes Bird and Wilson, 1994
W O 95/32288
DB#280 nematodes Bird and Wilson, 1994
W O 95/32288
Cat2 nematodes Niebel et al., 1995
aTub nematodes Aristizabal et al., 1996
sHSP nematodes Fenoll et al., 1997
Tswl2 nematodes Fenoll et al., 1997
Hs1 {prol nematodes WO 98/12335 - Jung
)
nsLTP viral, fungal, bacterialMolina & Garc'ia-Olmedo, 1993
RIP viral, fungal Tumer et al., 1997
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 Table 3
Table 3a
Average number of cysts and hatched juveniles obtained per plate, each plant
(containing four plants) was infected with 150 juveniles of H. schachtii (exp.
1 )
Mutant cysts per plate Juveniles per plate
Control 1 g 3230
DN-1 25 4350
DN-2 18 954
10 Table 3b
Average eggmass number and next generation juveniles obtained per plant (5
plants
per plate), each plant was infected with 100 juveniles of M. incognita (exp.
2)
Mutant eggmasses per plant Juveniles obtained per plant
Control 26 1729
DN-2 16 745
DN-3 18 209
Table 4
15 Cyst and eggmass numbers were counted per plant. In each plate two plants
were
grown and 100 juveniles per plate (50 juveniles/plant) of H. schachtii or M.
incognita
were used for infection. The average number of cysts or eggmasses and the
standard deviations {stdv) are shown in bold (exp. 3).
Lines Number of cysts per plant average std Number of eggmasses average stdv
v per plant
Control g 3 8 2 5 9 6.0 3.1 4 4 3 7 6 5 4.8 1.5
DN-1 6 5 3 0 7 10 5.2 3.4 5 2 3 5 3 1 3.2 1.6
DN-2 7 2 10 4 4 1 4.7 3.3 3 1 2.0 1.4
DN-3 0 0 0 0 0 0 0 2 0 0 0 0.5 1.0
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
56
Table 5
Line no. No. plantsNo. inoc No. Galls/ino
tips galls c tips
WT- 11 40 46 1,15
Desiree
S5-1 9 28 18 0,64
S5-2 7 26 26 1,00
S5-3 7 33 39 1,18
S5-4 6 27 29 1,07
S5-5 7 24 21 0,88
S11-1 9 16 14 0,88
S 11-2 6 39 45 1,15
S11-5 6 17 19 1,12
S 11-6 8 27 30 1,11
S11-9 7 34 31 0,91
S11-10 6 21 21 1,00
S11-12 7 39 36 0,92
S11-13 6 36 15 0,42
S11-14 7 31 35 1,13
S11-16 8 27 30 1,11
S11-17 8 47 50 1,06
S11-21 7 36 31 0,86
WT-Bintje 10 40 34 0.85
S8-4 7 12 13 1.08
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
57
REFERENCES
Abouzid et al., (1988) Mol. Gen. Genet. 212 : 252-258
Agrios GN, (1997). Plant Pathology, 4th edition, Academic Press; ISBN:
0120445646
Arion et al., (1988). Cell 55:371-378.
Aristizabal F, Serna L, Sanz-Alferrez S, Escobar C, Del Campo FF, Grundler F
and
Fenoll C (1996). Molecular analysis of nematode-inducible plant genes. gtn
International Congress on Plant-Microbe Interaction, Knoxville US B-29.
Banuett, F. (1992). Ustilago maydis, the delightful blight. Trends Genet. 8,
174-180.
Ballvora et al., (1995). Marker enrichment and high resolution map of the
segment of
potato chromosome VII harbouring the nematode resistance gene Grol., Mol. Gen.
Genet. 249:82-90.
Barthels, N., Karimi, M., Van Montagu, M., and Gheysen, G. (1994). Isolation
and
analysis of nematode-induced genes in Arabidopsis thaliana through in vivo ~3-
glucuronidase fusions. Med. Fac. Landbouww. Univ. Gent. 59/2b, 757-762.
Barthels, N., van der Lee, F.M., Klap, J., Goddijn, O.J.M., Karimi, M., Puzio,
P.,
Grundler, F.M.W., Ohl, S.A., Lindsey, K., Robertson, L., Robertson, W., Van
Montagu, M., Gheysen, G., and Sijmons, P. (1997). Regulatory sequences of
Arabidopsis drive reporter gene expression in nematode feeding structures. The
Plant cell 9, 2119-2134.
Bastos, N.C. (1985). A new pathotype of Crinipellis perniciosa (witches' broom
disease) on solanaceous hosts. Plant Pathol. 34, 306-312.
Bell et al., (1993). Plant Mol. Biol. 23:445-451.
Binarova et al., (1998). Plant J. 16: 691-707.
Bird, A.F. (1961). The ultrastructure and histochemistry of a nematode-induced
giant
cel I. J. Biophys. Biochem. Cytol. 11, 701-715.
Bird D, Wilson MA (1994) DNA sequence and expression analysis of root-knot
nematode-elicited giant cell transcripts, Mol. Plant-Microbe Interact., 7, 419-
42.
Bogre et al., (1997). PJant Physiol. 113: 841-852.
Bogre et al., (1998). The Planf Cell 11: 101-113.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
58
Burrows PR and de Waele D (1997). Engineering resistance against plant
parasitic
nematodes using anti-nematode genes, in Cellular and Molecular Aspects of
Plant-
Nematode Interactions, (Fenoll, C., Grundler, F. and Ohl, S., eds.), pp. 217-
236,
Kiuwer.
Braselton, J.P. (1995). Current status of the plasmidiophorids. Crit. Rev.
Microbiol.
21, 263-275.
Cai D et aL, (1997). Positional cloning of a gene for nematode resistance in
sugar
beet. Science 275:832-834.
Calderini et al (1998). J. Cell. Sci. 111: 3091-3100.
Calle, H., Cook, A.A., and Fernando, S.Y. (1982). Histology of witches' broom
caused in cacao (Theobroma cacao) by Crinipellis perniciosa. Phytopathology
72,
1479-1481.
Cid del Prado, V.I. and Maggenti, A.R. (1988). Description of Gracilacus
hamicaudata sp. n. (Nemata: Criconematidae) with biological and
histopathological
observations. Rev Nematol 11: 29-33.
Clarke, M.C., Wei, W., and Lindsey, K. (1992). High-frequency transformation
of
Arabidopsis thaliana by Agrobacterium tumefaciens. Plant Mol. Biol. Rep. 10,
178-
189.
Clough and Bent, Floral dip: a simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana, Plant J. 16 (1998), 735-743
Cohn, E. and Kaplan, D.T. 1983. Parasitic habits of Trophotylenchus
floridensis
(Tylenchulidae) and its taxonomic relationship to Tylenchulus semipenetrans
and
allied species. J Nematol 15: 514-523.
Cohen-Fix and Koshland, (1997). Curr. Opin. Cell Biol. 9:800-806.
Colasanti et al.,(1991 ). Proc. Natl. Acad. Sci. USA 88:3377-3381.
Cole, C.S. and Howard, H.W. 1958. Observations on giant cells in potato roots
infected with Heterodera rostochiensis. J Helminothol 32: 135-144.
Dahl et al., (1995). The Plant Cell7: 1847-1857.
Davies et al., J. Cell. Sci (Supply (1987) 7 : 95-107
Day AG; Bejarano ER; Buck KW; Burrell M; Lichtenstein CP (1991 ). Expression
of
an antisense viral gene in transgenic tobacco confers resistance to the DNA
virus
tomato golden mosaic virus. Proc Natl. Acad. Sci U S A, 88(15):6721-5
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
59
de Almeida Engler, J., De Vleesschauwer, V., Burssens, S., Celenza, J.L.,
Inze, D.,
Van Montagu, M., Engler, G., and Gheysen, G. (1999). Molecular markers and
cell
cycle inhibitors show cell cycle progression in nematode-induced galls and
syncytia.
The Plant cell 11 (5).
De Block, M. (1988). Genotype-independent leaf disc transformation of potato
(Solanum tuberosum) using Agrobacterium tumefaciens.
De Almeira Engler, J., De Vleesschauwer, V., Burssens, S., Celenza, J.L. Jr.,
Inze,
D., Van Montagu, M., Engler, G. and Gheysen, G. 1999. The use of molecular
markers and cell cycle inhibitors to analyze cell cycle progression in
nematode-
induced galls and syncytia. Plant Cell, in press, May 1999.
Dekhuijzen, H.M. (1980). The occurrence of free and bound cytokinins in
clubroots
and Plasmodiophora brassicae infected turnip cultures. Physiol. Plant. 49, 169-
176.
Dekhuijzen, H.M. and Overeem, J.C. (1971). The role of cytokinins in clubroot
formation. Physiol. Plant Pathol. 1, 151-162.
De Veylder L, Segers G, Glab N, Casteels P, Van Montagu M, Inze D, (1997). The
Arabidopsis CkslAt protein binds to the cyclin-dependent kinases Cdc2aAt and
cdc2At. FEBS Lett. 412, 446-452.
Doerner et a1.,1996. Nature 380:520-523.
Dropkin, V.H. and Nelson, P.E. (1960). The histopathology of root-knot
infections in
soybeans. Phytopathology 50, 442-447.
Elledge, (1996) Science B 274 1664-1672.
Endo, B.Y. (1964). Penetration and development of Heterodera glycines in
soybean
roots and related anatomical changes. Phytopathology 54, 79-88.
Endo, B.Y. (1971). Synthesis of nucleic acids at infection sites of soybean
roots
parasitized by Heterodera glycines. Phytopathology 61, 395-399.
Evans, H.C. (1980). Pleomorphism in Crinipellis perniciosa, causal agent of
witches'
broom disease of cocoa. Trans. Br. Myc. Soc. 74, 513-523.
Evans et al., (1983) Cell 33:389-396.
Fantes, P. (1979). Nature 279:428-430.
Federoff et al (1984) PNAS 81, 3825-3829.
Feiler H.S., Jacobs T., (1990}. Proc. Nat. Acad. Sci. 87:5397-5401.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 Fenoll C, Aristizabal F, San-Alferez S, Del Campo FF (1997) Regulation of
gene
expression in feeding sites. In: C. Fenoll, F.M.W. Grundler and S.A. Ohl
(Eds.),
Cellular and molecular aspects of plant-nematode interactions. Kluwer Academic
Publishers, Dordrecht, The Netherlands, pp. 133-149
Fesquet et al., {1993). EMBO J. 12:3111-3121.
10 Fitzmaurice WP; Lehman LJ; Nguyen LV; Thompson WF; Wernsman EA; Conkling
MA (1992). Development and characterization of a generalized gene tagging
system
for higher plants using an engineered maize transposon Ac. Plant Mol Biol,
20(2):177-98.
Francis D. & Halford N.G. Physiol. Plant 93:365-374, 1995.
15 Francis, D., Dudits, D., and Inze, D. (1998) Plant Cell Division, Portland
Press
Research Monograph X, Portland Press, London, see whole of contents
Frischmuth T and Stanley J {1998). Recombination between viral DNA and the
transgenic coat protein gene of African cassava mosaic geminivirus. J. General
Virology 79, 1265-1271.
20 Ganal MW et al (1995). Genetic mapping of a wide spectrum nematode
resistance
gene (Hero) against Globodera rostochiensis in tomato, Mol. Plant-Microbe
Interact.
8, 886-891.
Ganal MW, and Tanksley SD (1996). Recombination around the Tm2a and Mi
resistance genes in different crosses of Lycopersicon pervianum. , Theor.
Appl.
25 Genet. 92:101-108.
Gheysen, G., de Almeida Engler, J., and Van Montagu, M. 1997. Cell cycle
regulation in nematode feeding sites. In: C. Fenoll, F.M.W. Grundler and S.A.
Ohl
(Eds.), Cellular and molecular aspects of plant-nematode interactions. Kluwer
Academic Publishers, Dordrecht, The Netherlands, pp. 120-132.
30 Goodey, J.B. 1948. The galls caused by Anguillulina balsamophila (Thorne)
Goodey
on the leaves of Wyefhia amplexicaulis Nutt and Balsamorizha sagittata. J
Helminthol 22: 109-116.
Goverse A, de Almeida Engler J, Verhees J, van der Krol S, Helder J, Gheysen G
(1999). CELL CYCLE ACTIVATION BY PLANT PARASITIC NEMATODES, Plant
35 Molecular Biology, submitted
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
61
Griffiths, B.S. and Robertson, W.M. 1988. A quantitative study of changes
induced
by Xiphinema diversicaudatum in root-tip galls of strawberry and ryegrass.
Nematologica 34: 198-207.
Grimsley, Proc. Natl. Acad. Sci. USA 83 {1986), 3282-3286
Gurr SJ; McPherson MJ; Scollan C; Atkinson HJ; Bowles DJ (1991 ). Gene
expression in nematode-infected plant roots. Mol Gen Genet, 226(3):361-6
Hahn S. K., Terry, E. R. and Leuschner, K. (1980). Breeding cassava for
resistance
to cassava mosaic disease. Euphytica 29, 673-683.
Haseloff J; Siemering KR; Prasher DC; Hodge S (1997}. Removal of a cryptic
intron
and subcellular localization of green fluorescent protein are required to mark
transgenic Arabidopsis plants brightly. Proc Natl. Acad. Sci U S A, 94(6):2122-
7.
Hayles et al., {1986). EMBO J. 5: 3373-3379.
Hemerly, A.S., Ferreira, P.C.G., de Almeida Engler, J., Van Montagu, M.,
Engier, G.
and Inze, D. (1993). cdc2a expression in Arabidopsis thaliana is linked with
competence for cell division. Plant Cell 5: 1711-1723.
Hemerly, Engler, Bergounioux, Van Montgu, Engler, Inze, Ferreira, Dominant
negative mutants of the Cdc2 kinase uncouple cell division from iterative
plant
development, EMBO J. 14 (1995), 3925-3936
Hesse, ( 1969), Osterr. Bot. Z. 117, 411-425
Hesse M (1971 ) Haufigkeit and Mechanismen der durch gallbildeende Organismen
ausgeloster somatischen Polyploidiserung. Osterr. Bot. Z. 1179, 454-463.
Hirt et al., (1991 ). Proc. Natl. Acad. Sci. USA 82:820-823.
Hirt et al., (1992). The Plant Ce114: 1531-1538.
Hirt et al., (1993). Plant Journal4: 61-69.
Hochstrasser, (1998) Genes Dev. 12:901-907.
Holsters, M., Silva, B., Van Vliet, F., Genetello, C., De Block, M., Dhaese,
P.,
Depicker, A., Inze, D., Engler, G., Villaroel, R., Van Montagu, M., and
Schell, J.
(1980). The functional organization of the nopaline A. tumefaciens plasmid
pTiC58.
Plasmid 3, 212-230.
Holtorf, Ludwig-Miiller, Apel, Bohlmann, High-level expressing of a viscotoxin
in
Arabidopsis thaliana gives enhanced resistance against Plasmodiophora
brassicae.
Plant Mol. Biol. 36 (1998), 673-680
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
62
Hong Y; Saunders K; Hartley MR; Stanley J (1996) Resistance to geminivirus
infection by virus-induced expression of dianthin in transgenic plants.
Virology,
220(1 ):119-27.
Huang, C.S. and Maggenti, A.R. (1969a}. Mitotic aberrations and nuclear
changes of
developing giant cells in Vicia faba caused by root knot nematode, Meloidogyne
javanica. Phytopathology 59, 447-455.
Huang, C.S. and Maggenti, A.R. (1969b}. Wall modifications in developing giant
cells
of Vicia faba and Cucumis sativus induced by root knot nematode, Meloidogyne
javanica. Phytopathology 59, 931-937.
Huntley et al., (1998). Plant Mol. Biol. 37:155-169.
Hussey, (1989), Ann. Rev. Phytopathol. 27, 123-141
Jacobs et al. (1996). Mapping of resistance to the potato cyst nematode
Globodera
rostochiensis from the wild potato species Solanum vernei. , Mol. Breed. 2:51-
60.
John PCL (1981) in, The Cell Cycle, Cambridge University Press, Cambridge UK.
John PCL et al., (1989). Plant Cell 1:1185-1193.
John PCL et al, (1993}. In: Ormrod J.C., Francis, D. (eds) Molecular and Cell
biology
of the Plant Cell Cycle. pp. 9-34, Kluwer Academic Publishers, Dordrecht,
Netherlands.
John, P.C.L. (1998). Cytokinin stimulation of cell division: Essential signal
transduction is via Cdc25 phosphatase. J. Exp. Bot. 49 (suppl.}, 91.
Jones, M.G.K. and Northcote, D.H. (1972). Nematode-induced syncytium - a
multinucleate transfer cell. J. Cell Sci. 10, 789-809.
Jones, M.G.K. and Payne, H.L. (1978). Early stages of nematode-induced giant-
cell
formation in roots of Impatiens balsamina. J. Nematol. 10, 70-84.
Jones, M.G.K. (1981 ). The development and function of plant cells modified by
endoparasitic nematodes, in B.M. Zuckerman and R.A. Rohde (eds.), Plant
Parasitic
Nematodes, Vol. III, Academic Press, New York, pp. 255-279.
Jones and Dropkin, (1975). Cellular alterations induced in soybean roots by
three
endoparasitic nematodes. Physiological Plant Pathology 5, 119-124
Jones and Northcote, (1972), J. Cell Sci. 10, 789-809.
Jung C, Cai D, Kleine M (1998) Engineering nematode resistance in crop
species,
Trends Plant Sci. 3:266-271.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
63
Karimi, M., Van Poucke, K., Barthels, N., Van Montagu, M., and Gheysen, G.
(1998).
Introduction of nematode-inducibfe plant promoter-gus fusions in hairy roots
of
different plants. Med. Fac. Landbouww. Univ. Gent, in press
Kertbundit S; De Greve H; Deboeck F; Van Montagu M; Hernalsteens JP {1991) In
vivo random beta-glucuronidase gene fusions in Arabidopsis thaliana. Proc.
Natl.
Acad. Sci. USA, 88(12):5212-6.
Kheyr-Pour, Nucleic Acids Res. 19 (1991 ), 6763-6769
Koncz, C., and Schell, J. (1986). The promoter of TL-DNA gene 5 controls the
tissue-specific expression of chimeric genes carried by a novel type of
Agrobacterium binary vector. Mol. Gen. Genet. 204, 383-396.
Koncz C; Martini N; Mayerhofer R; Koncz-Kalman Z; Korber H; Redei GP; Schell J
(1989). High-frequency T-DNA-mediated gene tagging in plants. Proc Natl. Acad.
Sci
U S A, 86{21 ):8467-71.
Krek, {1998) Curr. Opin. Genet. Dev 8:36-42.
Kumagai A. and Dunphy W.G., (1991 ). Cell 64:904-914.
Kumar, A. (1991 ). Agrobacterium-mediated transformation of potato genotypes.
In
Methods I Molecular Biology, Vol. 44: Agrobacterium Protocols. K.M.A. Gartland
and
M.R. Davey (Eds.), Humana Press Inc., Totowa, NJ.
Labbe J-C et al, (1989) EMBO J. 8:3053-3058.
Lake R.S. & Salzman N.P. (1972). Biochemistry 11:4817-4825.
LaMondia, (1991 ), Plant Disease 75, 453-454; Omwega et al., 1990
Phytopathology
80, 745-748
Langhan T.A. (1978). Meth. Cell. Biol. 19:127-142.
Lawton K; Ward E; Payne G; Moyer M; Ryals J (1992) Acidic and basic class III
chitinase mRNA accumulation in response to TMV infection of tobacco. Plant Mol
Biol, 19(5):735-43.
Lazarowitz, S. G. (1992). Geminiviruses: Genome structure and gene function.
Critical Reviews in Plant Sciences 11 (4), 327-349.
Lebel E, Heifetz P, Thorne L, Uknes S, Ryals J, Ward E (1998) Functional
analysis
of regulatory sequences controlling PR-1 gene expression in Arabidopsis. Plant
J;16(2):223-33
Lee M.G. and Nurse P., Nature 327:31-35, 1987.
CA 02330550 2000-12-14
WO 99166055 PCT/EP99/04139
64
Lisztwan et al., (1998) EMBO J. 18:368-383.
Lundgren, et al., ( 1991 ). Cell 64: 1111-1122.
Magnusson, C. and Golinowski, W. (1991 ). Ultrastructural relationships of the
developing syncytium induced by Heterodera schachtii (Nematoda) in root
tissues of
rape. Can. J. Bot. 69, 44-52.
Manners JM; Penninckx IA; Vermaere K; Kazan K; Brown RL; Morgan A; Maclean
DJ; Curtis MD; Cammue BP; Broekaert WF (1998). The promoter of the plant
defensin gene PDF1.2 from Arabidopsis is systemically activated by fungal
pathogens and responds to methyl jasmonate but not to salicylic acid. Plant
Mol Biol,
38(6):1071-80.
Melchers LS, Apotheker-de Groot M, van der Knaap JA, Ponstein AS, Sela-
Buurlage
MB, Bol JF, Cornelissen BJ, van den Elzen PJ, Linthorst HJ (1994). A new class
of
tobacco chitinases homologous to bacterial exo-chitinases displays antifungal
activity. Plant J;5(4):469-80.
Mironov, V., De Veylder, L., Van Montagu, M. and Inze, D. (1999). Cyclin-
dependent
kinases and cell division in plants - The Nexus. Plant Cell 11, 509-521.
Molina A; Segura A; Garc~ia-Olmedo F (1993). Lipid transfer proteins (nsLTPs)
from
barley and maize leaves are potent inhibitors of bacterial and fungal plant
pathogens. FEES Lett, 316(2):119-22.
Morejohn LC et al. (1987). Planta 172, 252-264.
Morejohn, L.C., and Fosket, D.E. (1984). Taxol-induced rose microtubule
polymerization in vitro and its inhibition by colchicine. J. Cell Biol. 99,
141-147.
Mourgues F , Brisset MN, Chevreau E (1998) Strategies to improve plant
resistance
to bacterial disease through genetic engineering. TIBTECH, 16, 203-210.
Murray, A. and Kirschner, M. (1989). Science 246: 614-621.
Nasmyth K. (1993). Curr Opin. Cell Biol. 5:166-179.
Niebel A, Gheysen G, Van Montagu M. (1994). Plant-cyst nematode and plant-root
knot nematode interaction. Parasitol. Today 10, 424-430.
Niebel A, Heungens K, Barthels N, Inze D, Van Montagu M, Gheysen G (1995)
Characterization of a pathogen-induced potato catalase and its systemic
expression
upon nematode and bacterial infection. Mol Plant Microbe Interact 1995 May-
Jun;
8(3):371-8
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
5 Niewohner J, Salamini F. Gebhardt C (1995). Development of PCR assays
diagnostic for RFLP marker alleles closely linked to alleles Gro1 and H1
conferring
resistance to the root cyst nematode Globodera rostociensis in potato. Mol.
Breed.
1:65-78.
Noris E; Accotto GP; Tavazza R; Brunetti A; Crespi S; Tavazza M. {1996)
10 Resistance to tomato yellow leaf curl geminivirus in Nicotiana benthamiana
plants
transformed with a truncated viral C1 gene. Virology, 224(1):130-8
Norbury C. & Nurse P. Ann. Rev. Biochem. 61:441-470, 1992.
Nurse, P. (1990} Nature 344: 503-508.
Nurse P. and Bissett Y. (1981 ). Nature 292:558-560.
15 Ogawa, J.M., Zehr, E.I., Bird, G.W., Ritchie, D.F., Uriu, K. and Uyemoto
J.K. {eds.;
1995). Compendium of stone fruit diseases. The American Phytopathological
Society. APS Press.
Opperman, C. H., Taylor, C. G. & Conkling, M. A., (1994). Root-knot nematode-
directed expression of a plant root-specific gene. Science 263: 221-23.
20 Opperman CH, and Bird DM (1998). The soybean cyst nematode Heterodera
glycines: A genetic model system for the study of plant-parasitic nematodes.
Curr.
Opin. Plant Biol. 1, 342-346.
Ormrod, J.C., and Francis, D. (1993) Molecular and Cell Biology of the Plant
Cell
Cycle, Kluwer Academic Publishers, Dordrecht, Netherlands.
25 Pereira, J.L., Almeida, L.C.C.D. and Santos, S.M. (1996). Witches' broom
disease
of cocoa in Bahia: Attempts at eradication and containment. Crop Protection
15,
743-752.
Pines J. (1995) Biochem J. 308:697-711.
Plesse et al., (1998) in Plant Cell Division, (Portland Press Research
Monograph X,
30 D. Francis, D. Dudits and D. Inze, eds (London: Portland Press) 145-163.
Poon et al., (1993). EMBO J. 12:3113-3118, 1993.
Reed et al., (1985). Proc. Natl. Acad. Sci, USA 82:4055-4959.
Renaudin et al. (1996). Plant cyclins: A unified nomenclature for plant A- B-
and D-
type cylins based on sequence organisation. Plant Mol. Biol. 32, 1003-1018.
35 Rice, S.L., Leadbeater, B.S.C. and Stone, A.R. 1985. Changes in cell
structure in
roots of resistant potatoes parasitized by potato cyst nematodes. 1. Potatoes
with
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
66
resistance gene H1 derived from Solanum tuberosum ssp. andigena. Physiol Plant
Pathol 27: 219-234.
Riou-Khamlichi, C., Huntley, R., Jacqmard, A., Murray, J.A. (1999) Cytokinin
activation of Arabidopsis cell division through a D-type cyclin. Science 283,
1541-
1544.
Rohde, R.A. and McClure, M.A. (1975). Autoradiography of developing syncytia
in
cotton roots infected with Meloidogyne incognita. J. Nematol. 7, 64-69.
Rudgard, S.A. (1986). Witches' broom disease of cocoa in Rondonia, Brazil: pod
losses. Trop. Pest. Management 32, 24-26.
Ruiz-Medrano, Virology 253 (1999), 162-169
Russell P. & Nurse, P. (1986). Cel145:145-153.
Russell P. & Nurse P. (1987). Cel149:559-567.
Russell, P. & Nurse, P. (1987) Cel149:569-576.
Sano Y et al. (1998) Sequences of ten circular ss DNA components associated
with
the milk vetch dwarf virus genome. J. General Virology 79: 3111-3118.
Sasser JN, and Freckman DW, (1987). A world perspective on nematodolgy: the
role
of the society, in Vistas on Nematology, (J. Veech and D. Dickson, Eds.), pp.
7-14,
Society of Nematologists.
Sembdner, G. (1963). Anatomie der Heterodera rostochiensis Gallen an
Tomatenwurzeln. Nematologica 9: 55-64.
Schuler TH, Poppy GM, Kerry BR, Denholm I (1998) Insect resistant transgenic
plants. TIBTECH 16. 168-175.
Shorthouse JD, and Rohrfritsch O (1992). Biology of Insect-Induced Galls,
Oxford
University Press, New York.
Sijmons, P.C., Grundler, F.M.W., von Mende, N., Burrows, P.R., and Wyss, U.
(1991 ). Arabidopsis thaliana as a new model host for plant-parasitic
nematodes.
Plant J. 1, 245-254.
Sijmons et al. (1994), Ann. Rev. Phytopathol. 32, 235-259.
Soni, R., Carmichael, J.P., Shah, Z.H. and Murray, J.A. (1995). A family of
cyclin D
homologs from plants differentially controlled by growth regulators and
containing the
conserved retinoblastoma protein interaction motif. Plant Cell. 7, 85-103.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
67
Sorrell DA et al. (1999). Distinct cyclin D genes show mitotic accumulation or
constant levels of transcripts in tobacco Bright Yellow-2 cells. Plant
Physiol. 119,
343-351.
Starr, J.L. (1993). Dynamics of the nuclear complement of giant cells induced
by
Meloidogyne incognita. J. NematoG 25, 416-421.
Strittmatter G, Gheysen G, Gianinazzi-Pearson, Hahn K, Niebel A, Rohde W,
Tacke
E, (1996). Infections with various types of organisms stimulate transcription
from a
short promoter fragment of the potato gstl gene. Mol. Plant-Microbe Interact.
9, 68-
73.
Sun Y, Dilkes B, Zhang C, Dante R, Carneiro N, Lowe K, Jung R, Gordon-Kamm W,
and Larkins B (1999). Characterization of maize (Zea mays L.) Weei and its
activity
in developing endosperm. PNAS 1999 96: 4180-4185.
Sunter, G. and Bisaro, D. M. (1997). Virology 232 (2) 269-280.
Swenson et al., (1986) Cell 47:861-870.
Topping JF; Wei W; Lindsey K (1991). Functional tagging of regulatory elements
in
the plant genome. Development, 112(4):1009-19.
Torres-Pacheco, J. Gen. Virol. 74 (1993), 2226-2231
Tumer NE; Hwang DJ; Bonness M (1997) C-terminal deletion mutant of pokeweed
antiviral protein inhibits viral infection but does not depurinate host
ribosomes. Proc.
Natl. Acad. Sci. USA, 94(8):3866-71.
Valverde, M.E., Paredes-Lopez, O., Pataky, J.K. and Guevara-Lara, F. (1995).
Huitlacoche (Ustilago maydis) as a food source - biology, composition and
production. Crit. Rev. Food Sci. Nutr. 35, 191-229.
Vignutelli A, Wasternack C, Apel K, Bohlmann H. (1998) Systemic and local
induction of an Arabidopsis thionin gene by wounding and pathogens. Plant J;
14(3):285-95
Vovlas, N. and Troccoli, A. (1990). Histopathology of broad bean roots
infected by
the lesion nematode Pratylenchus penetrans. Nematol Mediter 18: 239-242.
Wang H, Fowke LC, Crosby WL (1997). A plant cyclin-dependent kinase inhibitor
gene. Nature 386: 451-452.
Ward et al. (1991) Plant Cell 3: 1085-1094.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
68
Wheeler, B.E.J. (1985). The growth of Crinipellis perniciosa in living and
dead cocoa
tissue. In: Developmental Biology of Higher Fungi (Moore, D., Casselton, L.A.,
Wood, D.A., Frankland, J.C., eds.), Cambridge University Press, Cambridge, UK.
pp. 103-116.
Wilson et al., (1998). PhysioG Planf 102: 532-538.
Wright, K.A. (1974). The feeding site and probable feeding mechanism of the
parasitic nematode Capillaria hepatica (Bancroft, 1893). Can J Zool 52: 1215-
1220.
Wyss, U., Lehmann, H. and Jank-Ladwig, R. (1980). Ultrastructure of modified
root-
tip cells in Ficus carica [figs], induced by the ectoparasitic nematode
Xiphinema
index. J Cell Sci 41: 193-208.
Xie et al., (1996). EMBO J. 15:4900-4908.
Yagoobi et al., (1995). Mapping a new nematode resistancelocus in Lycopersicon
esculentum, Theor. Appl. Genet. 91:457-464.
Young, C.W., and Hodas, S. (1964). Hydroxyurea: Inhibitory effect on DNA
metabolism. Science 146, 1172-1174.
Zeng et al., (1998). Nature 395: 607.
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: CropDesign N.V.
(B) STREET: Technologiepark 3
(C) CITY: Zwijnaarde-Gent
(D) STATE: none
(E) COUNTRY: Belgium
(F) POSTAL CODE (ZIP): 9052
(ii) TITLE OF INVENTION: Plant pathogen inducible control sequences operably
linked to cell cycle genes and the uses thereof
(iii) NUMBER OF SEQUENCES: 12
(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 N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
AATTTGGGTC TTGGTCGT 18
{2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
AGCAATCTTA AGAAGCTCTT 20
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99104139
2
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
CCCAAGCTTC TCCACTAGCC GTATTTTG 28
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
GCGCGTCGAC TTCCTATAAA GACTACCTCA 30
(2) INFORMATION FOR SEQ ID N0: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Glu Gly Thr Tyr Gly Val Val
1 5
(2) INFORMATION FOR SEQ ID N0: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:
Lys Leu Ala Asp Phe Gly Leu Ala Arg Se. Phe Gly Val Pro Leu Arg
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
3
1 5 10 15
Asn Tyr Thr His Glu Ile Val Thr Leu Trp Tyr
20 25
(2) INFORMATION FOR SEQ ID N0: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Val Thr Leu Leu Gln Asp Tyr Lys
1 5
(2) INFORMATION FOR SEQ ID N0: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Glu Gly Thr Tyr Gly Lys Val
1 5
(2) INFORMATION FOR SEQ ID N0: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Lys Leu Ala Asp Phe Gly Leu Ala Arg Ala Phe Gly Ile Pro Val Arg
1 5 10 15
Thr Phe Thr His Glu Val Val Thr Leu Trp Tyr
20 25
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
4
(2) INFORMATION FOR SEQ ID N0: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 10:
Lys Ile Ala Asp Leu Gly Leu Gly Arg Ala Phe Val Pro Leu Lys Ser
1 5 10 15
Tyr Thr His Glu Ile Val Thr Leu Trp Tyr
20 25
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 11:
Val Thr Ser Leu Pro Asp Tyr Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
Val Ser Thr Leu Arg Asp Trp Glu
1 5
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
CCCAAGCTTC TCCACTAGCC GTATTTTG 2g
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
GCGCGTCGAC TTCCTATAAA GACTACCTCA 30
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
Glu Gly Thr Tyr Gly Val Val
1 5
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:
Lys Leu Ala Asp Phe Gly Leu Ala Arg Ser Phe Gly Val Pro Leu Arg
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
6
1 5 10 15
Asn Tyr Thr His Glu Ile Val Thr Leu Trp Tyr
20 25
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
Val Thr Leu Leu Gln Asp Tyr Lys
1 5 ,
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 8:
Glu Gly Thr Tyr Gly Lys Val
1 5
(2) INFORMATION FOR SEQ ID N0: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
iD) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 9:
Lys Leu Ala Asp Phe Gly Leu Ala Arg Ala Phe Gly Ile Pro Val Arg
1 5 10 15
Thr Phe Thr His G1u Val Val Thr Leu Trp Tyr
20 25
CA 02330550 2000-12-14
WO 99/66055 PCT/EP99/04139
7
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 10:
Lys Ile Ala Asp Leu Gly Leu Gly Arg Ala Phe Val Pro Leu Lys Ser
1 5 10 15
Tyr Thr His Glu Ile Val Thr Leu Trp Tyr
20 25
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 11:
Val Thr Ser Leu Pro Asp Tyr Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
Val Ser Thr Leu Arg Asp Trp Glu
1 5
