WO1999066055A2

WO1999066055A2 - Plant pathogen inducible control sequences operably linked to cell cycle genes and the uses thereof

Info

Publication number: WO1999066055A2
Application number: PCT/EP1999/004139
Authority: WO
Inventors: Godlieve Gheysen; Vladimir Mironov; Dirk Gustaaf INZÉ; Franky Raymond Gerard Terras; Wim Van Camp; Ana Isabel Sanz Molinero
Original assignee: Cropdesign N.V.
Priority date: 1998-06-15
Filing date: 1999-06-15
Publication date: 1999-12-23
Also published as: WO1999066055A3; AU4772599A; CA2330550A1; AU757829B2; EP1088091A2

Abstract

Yield loss due to pathogenic, e.g., geminiviral or nematode, infection is a major problem in the cultivation of plants or, in particular, crops as such. Environmental concerns restrict the use of toxic compounds to combat said infectious agents. The current 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 or cell division of a plant cell.

Description

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 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. 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. avenae 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 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 fallax, 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. In 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 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: Gro1 (Ballvora et al., 1995; Jacobs et al., 1996), and H1 (Niewohner et al., 1995); in tomato: Mi-3 (Yagoobi et al., 1995) and Hero (Ganal et al., 1995) and Mi-1 (Ganal and Tanksley, 1996); and Hs1^pro'1 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 (interfering 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 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 Meloidogyne) 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 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 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 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 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 Begomovi ruses. Classification is based on genome structure (mono- or bipartite), natural vector (leaf hoppers or white fly species) and host range (mono or dicotyledonous). Mastrevirus are transmitted by leaf hoppers 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). 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://bioloav.anu.edu.au/Groups/MES/vide/genus005.htm) and that are known to cause economically important diseases affecting yield and quality of crops. 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 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.

Fungi

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 meristems (vegetative brooms) or of flower cushions. Flowers or pods can also be infected. Examples of fungi causing witches' broom are: Crinipellis 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 (Calle 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 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 host(s) 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 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 G₀-Gι 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 plumbaginifolia 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, 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, 1969). 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, Rhodococcus fascians, Pseudomonas savastanoi, Xanthomonas campestris pv citri. or Erwinia herbicola. 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 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 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 leakiness 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. 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 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 β-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 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 for 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: GO, Gapi (G1), DNA synthesis (S), Gap₂ (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). 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), Sid , Far1 , Rum1 , p21 , p27, p57, p16, p15, p18, p19 Pines, 1995; Elledge, 1996), p14 and p ARF; p13^suc1 or CKSIAt (Hayles et al., 1986, De Veylder et al., 1997) and nim-1 (Fantes, 1979; 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^cdc25 (Russell and Nurse, 1986; Kumagai and Dunphy, 1991 ; Bell et al., 1993; Elledge, 1996) and Pyp3 (Elledge, 1996); cdc2 protein kinase or p34^cdc2 (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^wee1( Russell and Nurse, 1986, 1987a, 1987b; Elledge, 1996; Sun et al., 1999), mikl (Lundgren et ai., 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^cdc28 (Reed et al., 1985; Nasmyth, 1993); p40^MO15 (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 cyclins, E2F and DP transcription factors, pocket proteins, CDC7/DBF4 kinase, CDC6, MCM2-7, Ore proteins, cdc45, components of SCF ubiquitin ligase, PCNA, DNA-polymerase. Other cell cycle control proteins that are involved in cyclin 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.

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.

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 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, Skp1 , Skp2, Rub1 , 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 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 (lntron-1 from the Shrunken gene of maize, for example) has been shown to increase expression levels by up to 100-fold. (Mait, 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. 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 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 G0/G1 transition (re-entry into the cell cycle) G1/S transition and S/G2/G1 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. The term "dominant 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 Tyr15 (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, ZmWeel 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 Tyr15 phosphorylation of CDC2 (Lundgren, Cell 64 (1991), 1111-1122). In Xenopus a MYT1 kinase has been identified that phosphorylates CDC2 at both Tyr15 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 plant 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 Mytl 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 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 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 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 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 bp 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 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 Borne, Mol. Gen. Genet. 243 (1994), 613-621 and in other sources. 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 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. Bi . 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, BioTechnology 10 (1992), 790-794; Zimmermann, Mol. Breeding 4 (1998), 369-379; Tavladoraki, Nature 366 (1993), 469-472) and dAbs (Benvenuto, Plant Mol. Biol. 17 (1991), 865-874) have been successfully expressed in Tobacco, Potato (Schouten, FEBS 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 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-omithine, 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, FEBS Lett. 389 (1996), 44-47) or β-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. 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 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 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 using wounded or enzyme-degraded immature embryos, or wounded or enzyme-degraded embryogenic callus and other methods 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. Nati. 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, Bio/Technology 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). 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 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 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 (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 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 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 (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 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. 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 transcriptional 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, "caps" 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. 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, cyclin 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 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 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, 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 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 transformation, Agrobacterium-meά\a\eό 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 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 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. A more detailed description of the invention, for the sake of clarity, is disclosed hereinafter.

The Figures 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). 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 μm 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 μm 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, Si 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. 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^"1 kanamycin monosulfate (Sigma) or 20 mg L^"1 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. P. 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 μL of X-gluc (20 mg in 1 mL of Λ/,Λ/-dimethyIformamide) 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 Millam (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. 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 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, Wl) and in pUC18, respectively. The site-directed mutagenesis was performed with the use of the ExSite PCR-based site-directed mutagenesis kit (Stratagene, 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 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 EcoRI digested (filled-in) plasmid pArm1-alll to produce transcriptional fusions of the Arml promoter and the mutant cDNA. The resulting expression cassettes composed of the Arml promoter, mutant CDC2aAt cDNAs and NOS polyadenylation site are generally named Arm1-cdcmut and were transferred as Xbal fragments into the Xbal site of the binary vector pGSC1704. ARM1 or AttOOOI is an A. thaliana line containing an in vivo β-glucuronidase fusion 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).

1.3 Arabidopsis transformation and expression analysis

The binary constructs were introduced in A. tumefaciens C58C1 Rif^R(pGV2260) by electroporation with subsequent selection for streptomycin/spectinomycin resistance. To confirm the presence and integrity of the plasmids the DNA extracted from the streptomycin/spectinomycin resistance clones was retransformed in E. Coli XL1-Blue and the plasmid DNA from the resultant clones was subjected to restriction digest analysis with BamHI/Kpnl. A. thaliana Col-0 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 Arml 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 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 P₀7₂8-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 lines have been generated.

In an initial screen, 18 independent putative transformed lines have been tested for nematode resistance. Therefore 6 to 9 clones (intemode segments and tops) of each of the 18 selected lines were grown individually on petridishes (145/20mm; greiner, Frickenhausen, Germany) containing 40ml 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). 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 FΛ 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 incognita 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, plantlets 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. Phenotvpic analysis of transgenic 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 esculemtum 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 bp 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. 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 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 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. 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 pBin 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 Funoal 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 10⁷ 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. 5.3 Phenotypic analysis of transgenic 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.

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 19 3230

DN-1 25 4350

DN-2 18 954

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 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 9 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 Table 5

Line no. No. plants No. inoc No. Galls/ino tips galls ctips

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

S11-2 6 39 45 1,15

S11-5 6 17 19 1,12

S11-6 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

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. 8^th 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 Grot, 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 β- 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 . 16: 691-707. Bird, A.F. (1961). The ultrastructure and histochemistry of a nematode-induced giant cell. d. 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). Plant Physiol. 113: 841-852. Bogre et al., (1998). The Plant Cell 11: 101-113. 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, Kluwer.

Braselton, J.P. (1995). Current status of the plasmidiophorids. Crit. Rev. Microbiol. 21 , 263-275.

Cai D er a/., (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 Cell 7: 1847-1857.

Davies et al., J. Cell. Sci (Suppl) (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 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 al.,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. 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. 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. 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. 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. 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. Goodey, J.B. 1948. The galls caused by Anguillulina balsamophila (Thorne) Goodey on the leaves of Wyethia 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 Molecular Biology, submitted 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.

Hemeriy, A.S., Ferreira, P.C.G., de Almeida Engler, J., Van Montagu, M., Engler, G. and Inze, D. (1993). cdc2a expression in Arabidopsis thaliana is linked with competence for cell division. Plant Cell 5: 1711-1723. Hemeriy, 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 Cell 4: 1531 -1538.

Hirt et al., (1993). Plant dournal 4: 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-Mϋller, Apel, Bohlmann, High-level expressing of a viscotoxin in Arabidopsis thaliana gives enhanced resistance against Plasmodiophora brassicae. Plant Mol. Biol. 36 (1998), 673-680 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. d. 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. Ill, 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. Karimi, M., Van Poucke, K., Barthels, N., Van Montagu, M., and Gheysen, G. (1998). Introduction of nematode-inducible 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; Hemalsteens 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 77:4817-4825. 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. 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. d. 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, Comelissen 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. FEBS 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 Niewόhner 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) 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. 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. 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. 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, 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. 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 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, d. 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). Cell 45Λ 45-153.

Russell P. & Nurse P. (1987). Cell 49:559-567.

Russell, P. & Nurse, P. (1987) Cell 49:569-576.

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. 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, d. Nematol. 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.) Wee1 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

Turner 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. 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 ai., (1998). Physiol. Plant 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.

Claims

1. A chimeric gene or recombinant DNA molecule comprising at least a plant pathogen inducible control sequence operably linked to a cell cycle gene.

2. The chimeric gene or recombinant DNA molecule of claim 1 , wherein said cell cycle gene is capable of modifying the cell cycle or cell division of a plant cell.

3. The chimeric gene or recombinant DNA molecule of claim 1 or 2, wherein said cell cycle gene is capable of modifying the cell cycle or cell division of a plant cell.

4. The chimeric gene or recombinant DNA molecule of any one of claims 1 to 3, wherein the control sequence is inducible either by a virus, a nematode, a fungus, a viroid, a bacterium, an insect or a parasitic plant.

5. The chimeric gene or recombinant DNA molecule of any one of claims 1 to 4, wherein said cell cycle gene is 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 a 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.

6. A vector comprising the chimeric gene or recombinant DNA molecule of any one of claims 1 to 5.

7. A host cell comprising the chimeric gene or recombinant DNA molecule of any one of claims 1 to 5 or the vector of claim 6.

8. A method for obtaining a plant with reduced susceptibility to pathogenic infection and/or spread thereof comprising the steps of transforming a recipient and/or plant cell with a chimeric gene or recombinant DNA molecule of any one of claims 1 to 5 or the vector of claim 6.

9. A plant cell comprising a chimeric gene or recombinant DNA molecule of any one of claims 1 to 5 or the vector of claim 6.

10. A plant comprising plant cells of claim 9.

11. Propagation material or harvestable parts of the plant of claim 10 such as leaves, flowers, seed, seedlings, roots, fruit, pollen or tubers comprising plant cells of claim 9.

12. A kit comprising the chimeric gene or recombinant DNA molecule of any one of claims 1 to 5 or the vector of claim 6.

13. 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.

14. Use of the chimeric gene or recombinant DNA molecule of any one of claims 1 to 5 or the vector of claim 6 in a tissue specific manner for the control of a plant pathogenic infection.

15. Use of a pathogen inducible promoter for the expression a cell cycle gene.

16. Use of a cell cycle gene or a pathogen inducible promoter for the construction of a chimeric gene or recombinant DNA molecule of any one of claims 1 to 5, the vector of claim 6 or for the generation of a host cell of claim 7 or a plant cell of claim 9.