EP1088091A2 - Sequences de regulation inductibles par des pathogenes de vegetaux, liees de maniere operationnelle a des genes du cycle cellulaire, et utilisation desdites sequences - Google Patents

Sequences de regulation inductibles par des pathogenes de vegetaux, liees de maniere operationnelle a des genes du cycle cellulaire, et utilisation desdites sequences

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EP1088091A2
EP1088091A2 EP99931073A EP99931073A EP1088091A2 EP 1088091 A2 EP1088091 A2 EP 1088091A2 EP 99931073 A EP99931073 A EP 99931073A EP 99931073 A EP99931073 A EP 99931073A EP 1088091 A2 EP1088091 A2 EP 1088091A2
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plant
gene
cell
cell cycle
plants
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Godlieve Gheysen
Vladimir Mironov
Dirk Gustaaf Inz
Franky Raymond Gerard Terras
Wim Van Camp
Ana Isabel Sanz Molinero
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CropDesign NV
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CropDesign NV
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    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
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    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • 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.
  • 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.
  • cytotoxins e.g. barnase/barstar
  • 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.
  • 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.
  • 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.
  • 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)
  • 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.
  • 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.
  • 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.
  • nematodes 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.
  • popular potato cultivars that are grown for consumption such as Bintje and Desiree
  • nematode-resistant potato varieties lack the desired traits of Bintje or Desiree.
  • traditional plant breeding is a slow process, requiring generally 5-10 years for the production of a new cultivar.
  • resistance genes usually operate against a very limited number of nematode races.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • a syncytium a large multinucleated hypertrophied cell generated by the fusion of neighboring protoplasts after partial cell wall dissolution.
  • 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).
  • 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 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.
  • Mastrevirus examples include: Maize Streak Virus (MSV), Digitaria Streak Virus (DSV) and Wheat Dwarf Virus (WDV).
  • Curtovirus examples include: Beet Curly Top Virus (BCTV) and Horseradish Curly Top Virus (HCTV).
  • Begomovirus examples 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).
  • BGMV Bean Golden Mosaic Virus
  • TPGV Texas Pepper Geminivirus
  • SqLCV Squash Leaf Curl Virus
  • AbMV Abutilon Mosaic Virus
  • AYMV Ageraturn Yellow Mosaic Virus
  • ACMV African Cassava Mosaic Virus
  • CSMV Chloris Striate Mosaic Virus
  • Tomato Yellow Leaf Curl Virus TYLCV
  • Tomato Golden Mosaic Virus TGMV
  • geminiviruses 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.
  • RNA transcripts 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • P. goeppertianum is a relatively minor disease (2.2% of plants are infected) but infected plants usually do not produce fruit.
  • 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.
  • 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.
  • 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.
  • 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 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).
  • cytokinin 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 G 0 -G ⁇ progression but might also be involved in G 2 -M transition (for review, see Mironov et al., 1999). Cytokinins also affect G 2 -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.
  • 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).
  • viruses which cause a cell proliferation in plants upon infection belong to the class of Reoviridae.
  • 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.
  • Nanoviruses 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).
  • MDV milk vetch dwarf virus
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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).
  • the root cortex promoter see, for example, the ToRD2 promoter - WO 97/05261).
  • 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.
  • control sequence or promoter is inducible by either a virus, a viroid, a nematode, a fungus, a bacterium, an insect or a parasitic plant.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • 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), Gap 2 (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.
  • 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).
  • 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).
  • 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
  • CKI cyclin dependent
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • eukaryotic cells 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).
  • 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.
  • a cell cycle gene is a gene such as a cyclin dependent kinase gene, a cyclin dependent kina
  • 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.
  • 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:
  • 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.
  • cyst nematodes 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
  • 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.
  • 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.
  • 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.
  • 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).
  • MIK1 acts cooperatively with the WEE1 protein kinase in the inhibitory Tyr15 phosphorylation of CDC2 (Lundgren, Cell 64 (1991), 1111-1122).
  • 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.
  • kinase 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.
  • Mytl kinase 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DNA molecules 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • RNA-DNA oligonucleotide a hybrid RNA-DNA oligonucleotide
  • chimeroplast a hybrid RNA-DNA oligonucleotide
  • a mutation or contains a heterologous region which is surrounded by the homologous regions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • said vector is a plant expression vector, preferably further comprising a selection marker for plants.
  • 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).
  • the chimeric promoters and recombinant genes of the invention can be reconstituted into liposomes for delivery to target cells.
  • 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.
  • hygro which confers resistance to hygromycin
  • Additional selectable genes 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.
  • mannose-6-phosphate isomerase which allows cells to utilize mannose
  • ODC ornithine decarboxylase
  • DFMO ornithine decarboxylase
  • 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.
  • 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 ⁇ -glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907).
  • luciferase PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121
  • green fluorescent protein Gerdes, FEBS Lett. 389 (1996), 44-47
  • ⁇ -glucuronidase Jefferson, EMBO J. 6 (1987), 3901-3907.
  • 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.
  • 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.
  • 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.
  • a chimeric gene, recombinant DNA molecule or vector of the invention into the genome of a plant, plant cell or plant tissue.
  • 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.
  • 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.
  • 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.
  • Agrobacterium tumefaciens is preferred in the method of the invention
  • other Agrobacterium strains such as Agrobacterium rhizogenes
  • 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 resulting transformed plant cell can then be used to regenerate a transformed plant in a manner known by a skilled person.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • resistance covers the range of protection from a delay to complete inhibition of disease development.
  • 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.
  • 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.
  • 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.
  • 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.
  • the above-described chimeric genes and recombinant DNA molecule can be used in combination with other transgenes that confer another phenotype to the plant.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • kit 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SCF complex e.g. Skp, cullin, F box protein
  • 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.
  • 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.
  • promoter refers herein to a "promoter” 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.
  • 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.
  • 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.
  • DNA molecule polynucleotide
  • DNA sequence DNA sequence
  • nucleic acid sequence nucleic acid sequence
  • nucleotide sequence refers 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.
  • 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 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-me ⁇ a ⁇ e ⁇ 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.
  • 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
  • Figure 2 shows the ARM1-alll plasmid containing a 3.7 kb ARM1 promoter fragment in pBluescript.
  • 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 ⁇ m nylon sieves (Falcon 2350 Cell Strainer; Becton Dickinson, Bedford, MA) submerged in sterile de- ionized water.
  • Potato cyst nematodes 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.
  • 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.
  • X-gluc Europa research products, Ely, U.K.
  • Jefferson (1987) 50 ⁇ L of X-gluc (20 mg in 1 mL of ⁇ /, ⁇ /-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.
  • DN 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):
  • 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.
  • the ARM1-alll plasmid contains a 3.7kb ARM1 promoter fragment in pBluescript (see Figure 2).
  • the binary constructs were introduced in A. tumefaciens C58C1 Rif R (pGV2260) by electroporation with subsequent selection for streptomycin/spectinomycin resistance.
  • A. tumefaciens C58C1 Rif R pGV2260
  • 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.
  • 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).
  • Potato transgenic lines (Desiree and Bintje) have been made harboring a P 0 7 2 8-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.
  • Constructs consisting of a CKI and a pathogen inducible promoter (nematode) (such as pAtt0728, pAtt1712), are transformed into tomato (see General Methods). About 100 transformants per construct are generated and analyzed by PCR for transgene integration. 50 transformants/construct are selected for seed production by self- pollination. The F ⁇ progeny are analyzed for stability of transgene expression (by RT- PCR) and for the number of transgene integration sites (by segregation analysis of the selectable marker). Based on these results, 20 lines/construct (10 plants/line) are selected for F 2 production. Amongst the F 2 populations, homozygous lines are selected for nematode resistance tests.
  • nematode pathogen inducible promoter
  • 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.
  • 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.
  • 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.
  • 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)
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Plasmodiophora brassicae of selected Arabidopsis lines transformed with the construct described above and of untransformed control lines can be assessed as described by Holtorf et al., (1998): A field isolate Plasmodiophora brassiscae is used to infect 10 days old A thaliana C24 seedlings grown in the greenhouse. They are then inoculated with 0.5 ml of a P. brassicae spore suspension in 50 mM potassium phosphate buffer (pH 5.5) containing 10 7 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
  • 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.
  • Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens. FEBS Lett, 316(2): 119-22.
  • 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

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Abstract

La perte de rendement due à une infestation par des pathogènes, par ex. des géminivirus ou des nématodes, constitue un problème majeur pour les plantes ou en particulier pour les cultures. Des impératifs en matière d'environnement restreignent l'utilisation de composés toxiques pour lutter contre lesdits agents infectieux. La présente invention concerne généralement des séquences de régulation inductibles par des pathogènes de plantes, telles que des promoteurs qui sont liés de manière opérationnelle à des gènes du cycle cellulaire et qui sont -en combinaison- capables de modifier le cycle cellulaire ou la division cellulaire d'une cellule végétale.
EP99931073A 1998-06-15 1999-06-15 Sequences de regulation inductibles par des pathogenes de vegetaux, liees de maniere operationnelle a des genes du cycle cellulaire, et utilisation desdites sequences Withdrawn EP1088091A2 (fr)

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WO1999066055A2 (fr) 1999-12-23

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