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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8237—Externally regulated expression systems
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • 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
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—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
  • C12N15/8279—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
  • 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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  • C12N2750/00011—Details
  • C12N2750/12011—Geminiviridae
  • C12N2750/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention relates to methods and materials for specifically expressing proteins and nucleic acid sequences in response to signals associated with endoparasitic nematode infection.
• Plant endoparasitic nematodes can infect a great variety of plant species, from annual crops to fruit trees (Agrios, 1988) . Since they constitute an important threat for many crop species, they are usually controlled by agrochemicals or through the use of natural resistance genes. Nematicides are expensive and often have a broad spectrum of toxicity which renders them potentially dangerous to animals and humans. In addition, resistance genes are not always available for a given crop, and even if they are, they are not always effective against nematodes under field conditions.
• Nematode Feeding Sites Endoparasitic nematodes are obligate biotrophs which induce specialized structures in the roots of parasitized plants, termed Nematode Feeding Sites or Nematode Feeding Structures
• NFSs are plant cells which have been modified by the nematode to fulfill its nutritional requirements : nematodes depend on NFS in an absolute way, since once the NFSs are formed, muscles in the nematode body degenerate and the animals become sedentary and linked to the NFS (reviewed in Sijmons et al . , 1994a) . If an NFS degenerates, the nematode cannot continue its development, and it dies before reproduction.
• Cyst nematode NFSs are multinucleated syncytia produced by the fusion of several adjacent cells; root- knot nematode NFSs are multinucleate giant cells formed through cell expansion and mitosis without cytokinesis (reviewed by Fenoll and del Campo, 1998)
• NFSs are formed from pre-existing root prevascular cells by a cell differentiation process triggered by the nematodes, thought to be via signal molecules present in their salivary secretions (reviewed by Hussey, 1989) .
• This differentiation process involves a first step in which DNA content increases by endorreduplication and, for root-knot nematodes, also by mitosis without cytokinesis. It has been demonstrated that cell cycle genes are activated by the nematode and that DNA synthesis occurs (reviewed by Gheysen et al , 1997), although the precise transcription factors responsible for NFS differentiation are unknown. Importantly many plant cells are known to enter endorreduplication pathways during differentiation (Melaragno et al . , 1993; Grafi and Larkins , 1995), thus this effect per se is not peculiar to NFS development.
• NFSs are potential targets for controlling nematode parasites by means of recombinant DNA technology to inactivate the NFS or render it unsuitable for nematodes.
• the extensive and/or strong expression of toxic or anti-NFS genes outside of the NFSs could produce undesirable effects on plant growth and development.
• NFS-inducible promoters with relatively high specificity, are desirable for such application.
• One method entails - probing the plant genome using sequences based on mRNA which is highly transcribed at infected tissues.
• a different method employs interposon tagging of the plant genome using promoterless GUS constructs.
• GVs Geminiviruses
• V- and C-sense Vector-sense and Complementary- sense
• GVs have been cloned and sequenced in the literature to date (over 60, including different strains) .
• TGMV Tomato Golden Mosaic Virus
• V sense promoters from GVs are particularly effective in promoting specific transcription of linked protein sequences in NFSs.
• novel techniques based on transient expression by microinjection in NFSs were employed novel techniques based on transient expression by microinjection in NFSs.
• V-sense promoters from monocot and dicot infecting viruses are induced in various plant species including Arabidopsis at NFSs at moderate to high levels, and at early stages after infection. Certain elements of such promoters have been identified as having a possible role in this inducibility .
• geminiviral promoters e.g. C-sense promoters, and V-sense promoters from bipartite B components
• V-sense promoters which in the virus are linked to the production of coat proteins, not of proteins related to cell growth or cell metabolism
• the inventors have demonstrated NFS specificity in that a construct carrying the V-sense promoter from the monocot infecting Wheat Dwarf Virus (WDV) has been shown to be expressed in Arabidopsis NFSs whereas the same construct is not expressed in a suspension of dividing cells from wheat. This suggests a crucial difference in terms of transcriptional activities between NFSs and ordinary dividing cells which had not previously been appreciated in the art. Nor had any link between GVs and endorreduplication processes been demonstrated.
• WDV Wheat Dwarf Virus
• the inventors have also investigated the transcription factors and transcriptional activators which can under certain circumstances be used to activate these promoters and have shown that at least two proteins (WDV Cl protein Seq ID No.4 in Figure 2 and human E2F-1) when expressed recombinantly in plant cells can enhance GV promoter-driven gene expression in plant cells. Indeed the E2F observations by the present inventors may have broad applicability for transcriptional activation of plant or plant viral promoters, as could the transactivation of heterologous GV V-sense promoters by GV Cl proteins.
• the inventors have provided methods and materials based on the above findings which can be used, inter alia , to control nematode infestation in plants, for instance via the overexpression at NFSs of specific genes which interfere with nematode development and thereby generate plants with reduced nematode susceptibility.
• a transcriptional inducer associated with a NFS for the activation of a GV V-sense promoter.
• the term "inducer” as applied to the promoter describes a trans acting molecule, or particular combination of molecules, which occur naturally in an NFS, and which can stimulate (activate) expression which is operably linked (under the control of) the GV V-sense inducible promoter.
• stimulation, activation or induction in relation to the present invention are all used broadly, and cover the situations in which the inducer is employed to activate a GV V-sense promoter in:
• “Operably linked” will generally imply joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
• NFS nematode feeding structure or site (e.g. multinucleated syncytia or giant cells formed through cell expansion and mitosis) at any stage of development, from initial feeding cell onwards, provided that differentiation has commenced .
• the NFS transcriptional inducer may be associated with any nematode-plant combination.
• root knot nematodes e.g. Meloidogyne incogni ta
• cyst nematodes e.g. Heterodera schachtii
• Lists of plant parasitic nematodes are given by Zuckerman et al (1971) (eds.) in: "Plant Parasitic Nematodes, Vol. I" pp 139-162, Pub. New York.
• the invention is applicable to any V-sense promoter capable of being so activated, e.g. those from monopartite viruses or the A component of bipartite viruses are particularly preferred.
• the transcriptional inducer may be one associated with a dicot NFS, and this is used to activate a monocot-infecting GV.
• Suitable monocot virus promoters include those from Wheat Dwarf Virus (WDV, see Collin et al . , 1996 - EMBL accession X02869) and MSV (Maize Streak Virus, see Fenoll et al . , 1988 -
• EMBL accession K02026) An example of a dicot virus promoter is that from Pepper Huasteco Virus (PHV) .
• PSV Pepper Huasteco Virus
• the promoter sequences from these viruses are shown in Figure 2, and are designated Seq ID Nos 1, 2 and 3 respectively.
• the use discussed above forms part of a method of inducing transcription from a nucleic acid comprising a nucleotide sequence operably linked to
• GV V-sense promoter the method comprising causing or allowing the exposure of the promoter to a transcriptional inducer molecule associated with an NFS.
• Such methods can be performed in vivo, both in planta and ex planta (e.g. using vectors in a suitable host cell) .
• the method may be employed in a test system for assessing the induction of a known, characterised, GV V-sense promoter with putative inducer molecules .
• it may be used to compare the response of different GV V-sense promoters, or modified versions thereof, with known inducers. Examples of both of these systems are set out hereinafter.
• the method is performed in vivo, i.e. the GV promoter is (stably) incorporated into a plant genome and the transcriptional inducer is present as part of the process by which an NFS is formed in that plant.
• the nucleic acid encoding the GV promoter and appropriate coding sequence may be present systemically in the plant, transcription is induced locally at an enhanced level, in response to the formation of the NFS.
• the invention provides a method for inducing NFS-specific transcription in a plant of a nucleotide sequence, said method comprising use of a GV V-sense promoter.
• a GV V-sense promoter particularly preferred is the use of a monocot GV V-sense promoter in a dicot plant.
• the "specificity" (or specific inducibility) in this and other aspects of the present invention may be manifest as localised transcriptional activation of the promoter only at NFSs, or transcription which is enhanced with respect to other regions of the plant .
• One embodiment of this aspect of the invention provides a method of reducing the susceptibility of the plant to nematode infection thereby obviating the need for extensive use of nematicides, by hindering the induction, development or correct functioning of the NFS. This can be achieved, for instance, by the localised transcription of a nematode control nucleotide sequence operatively linked to a GV V-sense promoter.
• nematode control nucleotide sequence is meant one which, when transcribed, will inhibit the parasitisation of a plant by an invading nematode. Broadly speaking this may be effected by either directly acting on the nematode, or by inhibiting the formation of the NFS upon which it depends. As described in more detail below, such sequences may encode proteins, but there is no absolute requirement for them to do so.
• sequences encoding nematicidal or cytotoxic protein or which block the expression of plant genes important for NFS or nematode development or functioning, or which cause toxin-mediated NFS-suicide.
• Targeting house-keeping genes (i.e. those required for cell viability) in the NFS may be preferred.
• the GV V-sense promoters may drive the transcription of nucleotide sequences (e.g. of genes or fragments of genes) encoding any of the following: nematicidal proteins; antisense mRNAs for genes important for NFS functions; proteins or fragments of proteins from plants or other organisms which interfere with the induction or maintenance of NFSs; ribozymes against mRNAs from genes required at NFS; sequences capable of co-suppressing these genes.
• nucleotide sequences e.g. of genes or fragments of genes
• nematode control sequences capable of any one or more of the following:
• Proteins suitable for use in this embodiment may include collagenases, snowdrop lectin or other lectins, inhibitors of nematode proteases, neuropeptide antagonists, antibodies to secretions (for an example of the successful use of antibodies to alter plant phenotype see e.g. Plant J. (1995) 8: page 745)
• RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene.
• Antisense technology is reviewed in Bourque, (1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.
• Protein targets known to be present at NFSs include: Lemmi9 from tomato; TobRB7 from tobacco; HMGRase, Histone 4A, cdc2aAt, cyclaAt, rhal from arabidopsis; ribulose phosphate epimerase (Favery et al, submitted) , genes corresponding to tagged sequences inducible by nematodes (Barthels et al, 1997) .
• Target proteins may be those which are crucial for NFS differentiation, expressed under the control of GV V-sense promoters e.g. E2F; RB; CDC2aAt
• GV V- sense promoters may be advantageous in this regard.
• the methods of the present invention preferably employ promoters derived from GVs which are not normally hosted by the plants in which the methods of the present invention are performed e.g. by using promoters from monocot infecting viruses in dicot plants.
• a promoter which is a variant of a GV V-sense promoter may be used in the various aspects of the invention.
• GV V sense promoter covers also these variant promoters.
• Changes which may be by way of base substitution, deletion, or addition, may be desirable for a number of reasons, including introducing or removing restriction endonuclease sequences, or altering the length, strength, or specificity of the promoter with respect to the native promoter. For instance it may be desirable to remove motifs (e.g. the GC boxes in MSV, see Fenoll et al . 1988, 1990) which may bind transcriptional factors and thereby reduce specificity.
• motifs e.g. the GC boxes in MSV, see Fenoll et al . 1988, 1990
• An Example of such a modified promoter sequence is presented in pGus208dgc, as described in Example 8.
• Variants may be prepared by those skilled in the art, for instance by site directed or random mutagenesis, or by direct synthesis.
• the variant nucleic acid is generated either directly or indirectly (e.g. via one or more amplification or replication steps) from an original nucleic acid having all or part of the native sequence (e.g. those shown in Seq ID Nos 1-3) .
• variants may include promoters which have been extended at the 3 ' or 5 ' terminus .
• fragments or other portions of the native sequences having the requisite activity as described above.
• restriction enzymes or nucleases may be used to digest a nucleic acid molecule, or mutagenesis may be employed, followed by an appropriate assay (for example using a reporter gene such as luciferase) to determine the sequence required.
• Portions may also be isolated by use of specific primers to amplify selected motifs or other elements, for instance by PCR.
• Fragments of interest include the conserved late element (CLE) disclosed in the Examples below, or, in the case of PHV, a 235 bp fragment .
• CLE conserved late element
• Similarity or identity between the variant and the native promoter from which it is derived may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J. Mol . Biol . 215: 403-10, which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman.
• Similarity or identity may be over the full-length of the relevant sequence, or may be over a part of it, preferably over a contiguous sequence of about or greater than 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300 or more nucleotide bases.
• the variant shares at least about 50%, or 60%, or
• filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes - 1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
• T m 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex
• the T m is 57°C.
• the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
• targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
• Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.
• suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS.
• suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.
• the level of activity may be quantified, for instance by using the transient expression methods discussed in more detail hereinafter, or methods analogous to those.
• Activity can then be assessed by the amount of mRNA produced by transcription from the promoter, or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter.
• the amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction.
• the reporter gene preferably encodes an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product.
• a detectable signal preferably a visually detectable signal, such as a coloured product.
• Many examples are known, including ⁇ -galactosidase, luciferase and green fluorescent protein (GFP) .
• GFP green fluorescent protein
• ⁇ -galactosidase activity may be assayed by production of blue colour on substrate, the assay being by eye or by use of a spectrophotometer to measure absorbance. Fluorescence, for example that produced as a result of GFP or luciferase activity, may be quantitated using a spectrophotometer.
• Radioactive assays may be used, for instance using chloramphenicol acetyltransferase, which may also be used in non-radioactive assays.
• the presence and/or amount of gene product resulting from expression from the reporter gene may be determined using a molecule able to bind the product, such as an antibody or fragment thereof .
• the binding molecule may be labelled directly or indirectly using any standard technique.
• one aspect of the present invention comprises a method for assessing the NFS-responsiveness of a variant promoter comprising the steps of:
• any suitable reporter/assay may be used for assessing variants and it should be appreciated that no particular choice is essential to, or a limitation of, the present invention.
• the compactness of the GV promoters renders them particularly suitable for fine dissection of discrete elements, and for carrying out deletion studies (e.g. to further improve NFS specificity) .
• Chimaeric promoters having the minimal elements or motifs responsible for NFS-inducible regulation, possibly in conjunction with other promoter sequences (e.g. taken from known plant promoters) form one part of the present invention.
• one apparently NFS-responsive element determined by the present inventors is the GV promoter CLE sequence, which makes a truncated 35S promoter active in NFSs.
• the localised inducer which gives rise to the specific transcriptional activation of the native or variant may derive directly from a nematode (e.g. from a salivary or other secretion) whereby, in nature, it acts directly on endogenous plant promoters to promote NFS formation.
• the transcriptional inducer may comprise a plant derived molecule, or battery of molecules, which does not occur elsewhere in the plant, but which plays a role in the development and differentiation of the NFS.
• an inducer may derive from neither the plant nor the nematode, but may be a heterologous molecule itself under the transcriptional control of an NFS-specific promoter.
• GV promoters may be activated, under certain circumstances, by the S-phase specific E2f transcription factor.
• E2F binding sites appears not to be a general feature of all GV V- sense promoters, or even of the subgroup I geminiviruses. Nor do E2F consensus sites appear to be widespread in plant promoter sequences. Thus their presence in WDV and MSV is particularly unexpected.
• the findings regarding Cl and E2F may be exploited to increase the activation of the NFS- specific transcription.
• the inventors have provided a method for enhancing the induction of NFS-specific transcription in a plant of a first nucleotide sequence operably linked to an NFS-induced promoter, said method comprising use of an NFS-induced promoter operably linked to a second nucleotide sequence encoding a transcriptional inducer of the NFS-induced promoter.
• This approach provides a "two component" system in which, for instance, a GV-V-sense promoter-anti-nematode fusion, is over- activated by a second fusion expressing an inducer (transcription factor, or transcriptional activator) of that promoter, under the control of that promoter.
• inducer transcription factor, or transcriptional activator
• the first and second promoters will generally be the same.
• GV-V-sense promoters or variants thereof in order to avoid undesirable expression of components.
• this approach will effectively amplify specificity, particularly if the first promoter is a variant promoter containing multiple transcription factor-binding sites while the second is a native GV-V-sense promoter.
• the inducers will be selected from agents which are not naturally occurring in the host plant, for instance WDV Cl protein and E2F-1 (or analogs thereof capable of activating the promoter) .
• WDV Cl protein and E2F-1 or analogs thereof capable of activating the promoter.
• the Examples below also show the use of mutant, modified, inducers based on Cl (see Example 9) .
• GV-sense promoters are activated by E2F may be utilised by using the V-sense promoter (or part of it fused to a core mammalian promoter) to drive gene expression in animal cells in which E2F activity is high (such as tumour cells) of control sequences with the purpose of diagnosing them or ⁇ ablating them.
• nucleic acid molecule comprising a GV-V-sense promoter (or variant thereof e.g. a chimaeric promoter) operably linked to a sequence which it may be desired to express in a NFS-specific manner e.g. a control nucleotide sequence.
• the various promoters and control sequences may be selected from any of those discussed above.
• constructs embodying the "two component system” are also included.
• Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid) .
• a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.
• Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term “isolated” encompasses all of these possibilities.
• the nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e.g. using an automated synthesiser.
• the nucleic acid may be in the form of a recombinant vector, -e.g. a replicable vector.
• Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform a prokaryotic or eukaryotic host (or shuttle between the two) either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) .
• Suitable vectors, and appropriate host cells can be readily chosen or constructed, containing appropriate regulatory sequences, including terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular
• nucleic acid constructs and vectors which operate in plants are particularly of interest in the present context.
• selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to antibiotics or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate) .
• antibiotics or herbicides e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate
• the present invention also provides methods comprising introduction of such a construct into a plant cell.
• a host cell containing a heterologous construct according to the present invention especially a plant or a microbial cell.
• heterologous is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention.
• the host cell e.g. plant cell
• the construct is preferably transformed by the construct, which is to say that the construct becomes established within the cell, altering one or more of the cell's characteristics and hence phenotype e.g. with respect to nematode resistance .
• Nucleic acid can be transformed into plant cells using any suitable technology, such as a disarmed Ti -plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718 , NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al .
• Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically "relevant monocot plants (see e.g. Hiei et al . (1994) The Plant Journal 6, 271-282)). Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium alone is inefficient or ineffective.
• a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A- 486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233 ) .
• a further aspect of the present invention provides a method of transforming a plant cell involving introduction of a construct as described above into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce a nucleic acid according to the present invention into the genome.
• the invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention (e.g comprising the GV-V sense promoter plus nematode control sequence) especially a plant or a microbial cell.
• a host cell transformed with nucleic acid or a vector according to the present invention e.g comprising the GV-V sense promoter plus nematode control sequence
• the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. There may be more than one heterologous nucleotide sequence per haploid genome .
• a plant may be " regenerated, e.g. from single cells, callus tissue, meristematic cell clusters, somatic embryos, or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al . , Cell Cul ture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications , Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.
• Regenerated plants which include a plant cell according to the invention are also provided, along with any part thereof, seed, somatic embryo, clone, selfed or hybrid progeny and descendants and any part of these which includes the transformed cell.
• the invention particularly provides a plant propagule from such plants, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on.
• a plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders' Rights. It is noted that a plant need not be considered a "plant variety” simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.
• the invention further provides a method of altering the nematode- resistance of a plant, the method comprising use of the nucleic acid constructs or vectors described above, optionally preceded by the earlier step of introducing them into a cell of the ptant or an ancestor thereof .
• plants according to the present invention will have enhanced nematode resistance by virtue of the inducible nematode control sequences therein.
• Resistance in this context means that the plants show a reduced susceptibility to parasitisation by a nematode as compared to a control plant. This may be assessed, for instance, by standard techniques (see e.g. Lamondia (1991), Plant Disease 75, 453-454; Omwega et al (1990) Phytopathol 80, 745-748). Those skilled in the art will appreciate that even embodiments of the invention which do not provide absolute resistance to nematode colonisation or predation, but instead simply reduce its severity, may still be commercially and practically beneficial.
• sequences e.g. anti-nematode or cytotoxic proteins
• sequences may produce deleterious or other undesirable effects through being expressed elsewhere in the plant (e.g. at edible parts, or under special untested environmental conditions) or in other plant species (for instance following cross pollination in the field, for example) .
• This may enhance the value of the plants from a regulatory or public perception perspective.
• Hpall restriction site is shown in lower case (correspond to 2610 and 321 positions) .
• nucleotides different to those of the WDV genome (x02869) from genebank database. They are:
• nucleotides and amino-acids which are different from the published in genebank database (X02869) . They are:
• Figure 6 This shows potential E2F-binding sites in the WDV Large Intergenic Region.
• A Diagram of the LIR from WDV (X02869) . Coordinates in the circular genome are indicated by arrowheads. The hairpin loop conserved by all geminiviruses and implicated in replication is marked, so as the beginning of ORFs VI and Cl which correspond to the virion- and complementary-sense transcription units, respectively. The orientation of the two E2F-like binding sequences, WDV-1 and WDV-2, are indicated by arrows, and their actual sequences shown below.
• A Diagram of the reporter plasmids used in the One-HybridTM system. Oligonucleotides containing three tandem copies of WDV1 were inserted upstream form the promoters of the reporter genes lacZ and HIS3 . Integration of both vectors into the yeast genome yielded the reporter yeast strain YSCilO.
• E2F-1 Interaction of E2F-1 with the WDV sequence.
• YSCilO was transformed with plasmids containing different fusions to the GAL4 activation domain, and checked for growth on selective medium and for ⁇ -galactosidase activity.
• Transformants carrying a fusion to the human E2F-1 protein pGAD424XhoIE2F-l
• pGAD424XhoIE2F-l grew on the selective medium and showed blue colour in a filter lift assay, indicating an interaction between human E2F-1 and WDVl.
• a yeast reporter strain (see Example 4) was transformed with a plasmid expressing a fusion of the GAL4 DNA-binding domain to a maize RB (pAS/zRb (RV-C) ) , plus a plasmid containing a fusion of the GAL4 activation domain the human E2F-1 (pGAD424XhoIE2F-l) .
• Figure 8 This shows putative E2F binding sites in GVs.
• Figure 10 This shows the preparation of pJIT39 (Examples 3 and 6) .
• the open reading frames of WDV are shaded in plain grey (Cl and C2) or striped (VI and V2) .
• the tag at the end of the C2 coding region is indicated as a black rectangle.
• the pBluescript vector sequences are in bold lines.
• the construction of the plasmid is detailed in the text.
• the WDV open reading frames are in grey or striped as above.
• Bold lines either side of the insert are pSELECTTM.
• the coding sequence of WDV Cl protein was cloned under the control of a double 35S promoter (grey rectangles) .
• the WDV genomic sequence is indicated as a dashed rectangle, with the intron region shaded in black.
• the Cl STOP codon is also indicated.
• the whole sequence of pJIT163BgIII is not indicated but only represented as a bold line on both sides of the insert.
• FIG. 13 A This shows the MSV LIR in pGUS208 and flanking regions in pG208. The positions where the mutagenesis oligonucleotides bind are shown by arrows (see Example 8) .
• the arrows mark the positions where the oligonucleotides OM1, OM2 , OM3 and OM4 hybridise.
• the position where the mutation was introduced to make elm is shown in bold and lowercase next to oligonucleotides OM2 and OM3 (G to A in the DNA sequence, E to K in the protein sequence) .
• Hindlll (AAGCTT) and ⁇ VdeJ (CATATG) sites are boxed in grey.
• nucleotides and aminoacids (a. a.) different from those published in genebank database (X02869) . They are :
• Example 1 Nematode induction of PHV promoter and chimaeric constructs in transgenic tobacco plants
• Promoter-GUS fusion 5 dpi 10 dpi 15 dpi 20 dpi
• a construct harbouring a truncated -90 35S promoter flanked by a dimer of the CLE element was very responsive to nematodes at very short infection times. It also showed a temporally-related expression in some organs in non-infected plants, which did not appear to be associated with any particular organ or cell type.
• the CLE is a conserved element which occurs in 20 or 30 different GVs. For instance it occurs (in one or more copies) in the C subregion (from the end of the loop to the beginning of rightward transcription) of the bipartite geminiviruses (subgroup III) isolates from the old world in all members except in the A component of mungbean yellow mosaic virus (MYMV) . It is present too in isolates from the New World like TGMV, SqLCV, PJV and PHV (see Arg ⁇ ello-Astorga et al . , 1994) .
• the CLE can, in some contexts, confer strong and early nematode inducibility to a -90 35S promoter .
• Example 2 Transient expression of GV promoters at NFSs in Arabidopsis Transient expression of MSV and WDV promoter-GUS fusions were tested in NFSs using a novel microinjection technique based on that developed in NFSs induced by cyst nematodes in Arabidopsis roots (Bockenhoff and Grundler, 1994) .
• the plasmids are shown in Figure 4.
• the plasmids pMOG969 (Ohl et al . , 1997) and HMG1-GUS (Lumbreras, 1995) were controls.
• pWDV4 plasmids are based on WDV and are disclosed in Collin et al . (1996) .
• pWDV4iGUS cannot produce Cl and does not give GUS activity, or very little, in wheat cells.
• Plasmid pWDV3GUS can produce Cl but cannot replicate and does not give GUS activity in wheat cells either (Hofer et al, 1992).
• PGUS207 and pGUS208 are based on MSV and can only produce truncated versions of Cl. They were prepared by cloning the Hind III/Xba I fragment from pCF208 and pCF207 (Fenoll et al . , 1988) in the plasmid pGUSl from Plant Genetic Systems in the sites Hindlll, Sal I, blunt-ended by fill-in reaction in the sites Xbal and Sail, 5' from the coding sequence of the GUS gene.
• Plasmid DNA for microinjections was purified by using a commercial kit (Quiagen-tip 100), the DNA concentration adjusted to 0.7 ⁇ M and lucifer yellow 2 mM was added to visually follow the microinjection. From this solution, 10 to 100 pL were microinjected using a glass needle with a pore diameter of 0.2 to 0.5 micrometers into 8 days old NFSs, and roots were stained for GUS 24 to 48 hors later. GUS expression, when found, was restricted to the syncytium. The results are summarized in Table 2:
• a control promoter known to be nematode-inducible (pMOG969 - see Ohl et al . , 1997) was also positive (albeit at a low level) while other constructs gave negative results (such as HMG1-GUS, which is induced by root-knot nematodes but not cyst nematodes: unpublished data plus Lumbreras , 1995), showing that GUS expression depended only on particular constructs, and was not an artefact for the microinjection procedure.
• pWDV3GUS when bombarded in wheat cells (Hofer et al . , 1992; Collin et al, 1996) did not show GUS expression, in spite of expressing the Cl protein; the fact that this construct is active in NFS indicates a crucial difference between dividing cells and NFS in the sense that Arabidopsis NFSs must have a transcriptional activity required for WDV V-sense expression that is not present in wheat dividing cells.
• pWDV4iGUS was expressed at only slightly higher than background levels in wheat cells (Collin et al, 1996) but gave clear induction in NFSs.
• the WDV promoter appears to be particularly NFS specific in that it is not expressed in dividing cells which are synthesizing DNA, which are likely to be the cells which most resemble NFSs in terms of transcriptional activity.
• GUS-fusions based on the promoter from MSV which infects monocots are expressed in a dicot plant in NFSs induced by cyst nematodes.
• pWDV-based constructs which have longer regions of the virus genome and code for viral proteins, can also be expressed in NFSs in a non-host plant.
• the V-sense WDV promoter is expressed in NFS, but not in dividing wheat cells, indicating that is not simply a promoter active in all cells in active proliferation and adding value to its application as an NFS-specific promoter.
• Example 3 Preparation of transgenic nematode-resistant plants using GV V-sense promoters and variants thereof General approach
• Transgenes can be stably introduced in different plant species by conventional methods.
• the gene fusions to be transformed contain a selected GV V-sense promoter driving gene expression specifically at NFSs, the gene being capable of reducing NFS viability and hence inhibiting nematode development, leading to increased nematode resistance in the transgenic plant.
• a GV V-sense promoter such as the WDV intergenic region present in pJIT39, and also a modified (variant) version of this promoter in which the original unique WDV-1 is replaced by a tetramer of the WDV-1 element (an E2F-1 binding site, see below) .
• the activity of the promoters is tested by fusing them to the GUS reporter gene sequence, introducing the fusion into a binary vector and transforming Arabidopsis plants via Agrobacterium.
• Transgenic lines are selected as TRANSGENIC REPORTER LINES (see also Example 7) and the GUS expression pattern in non-infected plants and in nematode infected plants will be studied.
• the GV promoter may be used to drive the expression of a cytotoxin, such as the RIP dianthin, with the aim of producing cell death at NFSs.
• a cytotoxin such as the RIP dianthin
• These protein have been shown to produce death in a cell- specific manner in other plant systems (for an example, see dianthin 30 used by Hong et al . , 1996) .
• Other proteins or parts of proteins with non-species specific cytotoxic effects may also be considered.
• the GV promoters may be used to drive the expression of antisense RNA for Arabidopsis genes which are expressed at NFSs, such as the HMG1 gene coding region, the sequence being obtained from the cDNA (Caelles et al . , 1989 Plant Mol Biol 13: 627-638).
• the HMG1 gene is highly induced by nematodes from very early during infection until completion of nematode development (unpublished) , and the HMG1 protein may be necessary for NFS development (see Fenoll et al . , 1997 for a discussion) . It catalyzes the first step in the synthesis of isoprenoid compounds, such as sterols and other lipids, essential for membrane growth and therefore essential for NFS growth and development .
• fusions of appropriate sense or anti-sense DNA coding sequences will be made by conventional PCR techniques to the promoter sequence in pJIT39.
• the fusions will be introduced in a binary vector, such as pBinl9 or a similar plasmid, and transformed into Agrobacterium tumefaciens, which will mobilize the constructs into Arabidopsis plants.
• Agrobacterium tumefaciens which will mobilize the constructs into Arabidopsis plants.
• presence of the anti-nematode gene fusions will be confirmed by conventional methods, such as PCR.
• Several independent transgenic lines may be selected for further analysis e.g. observation of the whole plant life cycle, and nematode-susceptibility tests, to determine the relative decrease in susceptibility of the transgenic plants (PRIMARY
• RESISTANT PLANTS - see Example 7 as compared to wt Arabidopsis plants.
• the nematode species which will be preferentially tested are Meloidogyne spp . and Heterodera schachtii . Naturally the above approach will be readily transferable to other plant species (particularly dicot plants) by those skilled in the art.
• WDV and MSV (natural and modified) promoters are set out below:
• the pGreen vector is based on the general cloning vector pBluescript (Alting-Mees and Short, (1989) Nucl . Acids Res. 17, 9494) and therefore contains a colEl ori for replication in E. coli .
• This plasmid' s ampicillin resistance gene (encoding kanamycin resistance)
• the pSa ori was inserted, the fl ori and Lac Z' region deleted and a Bglll site was left for the introduction of a T-DNA cassette.
• the T-DNA cassette in the case of pGreenOOOO is a 813bp Bglll fragment including the pBluescript SKII LacZ' and multiple cloning site, synthetic LB and B sequences, derived from the border sequences of PtT37 and with an additional T-DNA transfer enhancer ("overdrive") motif immediately adjacent and external to the RB (Slightom et al . , (1985) EMBO J. 3, 3069-3077; Van Haaren et al, (1988) Plant Mol. Biol . 11, 773-781.).
• the basic pGreen vector contains no selectable marker or reporter genes for plant transformation. Internal to the T-DNA (LB and RB respectively) are unique Hpal and Stul sites for the insertion of selectable marker or reporter gene cassettes. Four selectable marker genes and two reporter genes have been modified by site directed mutagenesis, to remove all the restriction sites which would have been duplicated in the pGreen multiple cloning site. This includes the aph3'II (kan; resistance to kanamychin; Bevan et al . , 1983) gene. The aph3'II gene does not contain the mutation that can affect its function as a selectable marker gene in some plant species (Yenifsky et al . , (1990) Proc . Natl . Acad.
• the function of the enzymes has not been affected by the DNA sequence changes introduced.
• the aph3'II gene coding sequence was fused to the nopaline synthase (nos) promoter-terminator and all extraneous sites at cloning junctions were removed.
• pGreen 0029 was constructed by taking an EcoRV fragment harbouring nos: aph3'II (kansoph II); nos terminator into the Hpa I site of pGreen 0000 at the left border (see Figure 5A) using standard techniques. Nomenclature, from 1 to 728, was based on the use of a computer generated matrix to assign numbers for all the possible 35S and nos -containing cassettes cloned into the LB and/or RB cloning sites of pGreenOOOO.
• Kpnl and the larger plasmid DNA band was separated by agarose gel electrophoresis and eluted from the gel.
• the pV fragment in the M13mpl8 phage was cut with Pstl, T4 polymerase I treated and then cut with Kpnl to make a blunt end Kpnl fragment which could be cloned into the Smal and Kpnl sites respectively of pJIT75.
• the end-result is pJIT39 (see 2 in Figure 10) in which the pV promoter effectively replaces the CaMV 35S promoter.
• Example 2 The preparation of pGUS208 is described in Example 2, and Figure 4.
• the modified DGC version is described in more detail in Example 8 and Figure 13.
• a 3708 bp HindiII -Xbal LIR MSV-GUS-ocs polyA fragment from pGUS208 was inserted into the Kpnl-BamHI site in pGreen 0029 creating pGreen 0029:GUS208, using standard techniques . Plasmid handling and copy number in Agrobacterium
• pGreen plasmid requires the function of the RepA of pSoup (pJICSa_rep) to be maintained.
• pGreen contains no mobilisation function and so the plasmid is introduced into
• a mixture of the pGreen plasmid and the pSoup can be used in a mixed electroporation.
• selection for co-transformed Agrobacterium can be achieved by selection on kanamycin-containing medium only, since pGreen cannot replicate in Agrobacterium without pSoup being coresident.
• electrocompetent Agrobacterium containing pSoup can be generated and re-electroporated with pGreen.
• a tumefaciens strain AGL-1 supports pGreen replication provided that pSoup is also present.
• tumefaciens strain AGL-1 harbouring pSoup and pGreen 0029:JIT39 OR pGreen 0029:GUS208 OR pGreen 0029:G208DGC can be transformed into tobacco using standard protocols, for example as generally described by Guerineau F., et al . , (1990) Plant Molecular Biology 15, 127-136.
• HindiII digests give plant DNA-TDNA border fragments of a minimum size of 5.5 Kb. Each band observed represented an independent integration event. 7 positive lines were observed for GUS208 transgenic lines ranging from single locus to >9 loci when probed with a GUS fragment .
• the GUS probe was prepared form a Smal 1.8Kb fragment eluted from PJIT166 (Guerineau et al . (1992) Plant Mol. Biol . 18, pp 815-818) using standard procedures. In all lines these fragments were greater than the minimum size and therefore were likely to be intact genes.
• GUSDGC lines 5 lines were identified ranging from a single locus to >5 loci. The same criteria apply as for GUS 208 lines.
• Transgenic tobaco seeds were provided as described above. In vitro-grown plants (1 to 3 weeks old) were stained for GUS activity. GUS activity was determined in whole plants directly in the Petri dishes as described by Jefferson (1987) .
• Stems and leaves Weak expression in vascular tissue, mainly in stems. It is the stronger expresser, as compared with pG208 and pG208dgc.
• Stems and leaves Very weak in vascular tissue, mainly in stems. Occasional weak expression in stems at the sites of adventitious root emergence, where cortical tissue breaks as the roots emerge. pG208dgc (3 lines)
• Stems and leaves Very weak in vascular tissue, mainly in stems.
• transgenic tobacco plants were selected on kanamycin then transferred to pots containing sterilised soil and grown at 25°C/18°C (day/night) under a 16h light photoperiod for 1 week.
• Nematode inoculation was carried out as follows. A suspension containing 1000 juveniles per ml of tap water was carefully pipetted through a wide-mouth pipette to minimise nematode shearing. A total of 0.5 ml of nematode suspension per plant (500 juveniles) were inoculated at a 1 cm depth in the soil immediately adjacent to the stem. After inoculation, plants were grown for 6 days, then carefully removed from the pots. The roots were washed with tap water to eliminate soil particles, and stained for GUS activity as described previously. After 12h staining, plant material was de-stained and observed under a binocular microscope (Leika MZ6) .
• pG208dgc Clear GUS staining in the galls was observed in one line of the pG208dgc construct. In some cases this was clearly located on the inside of the gall, suggesting that the NFS itself was expressing the transgene as expected. On other occasions, GUS expression seemed broader in the gall, perhaps affecting the vasculature and the other tissues forming the gall.
• Example 4 Regulation of GV V-sense promoters by cellular and viral proteins
• the large intergenic region of WDV (LIR) was studied for E2F binding sites . This region contains the bidirectional promoter of the virus.
• Two potential E2F-binding sites were found on the complementary strand between positions 2742-2730 (WDVl) and 205- 192 (WDV2) ( Figure 6A) .
• a comparison with functionally recognised E2F binding sites showed that the WDV sequences are not identical to the consensus sequence described for mammalian promoters ( Figure 6B) , but when comparison was made with the preferred binding site for E2F-1 (class I) , the viral sequences showed only a 1 (WDVl) or 2 (WDV2) base pair mismatch. Since these changes in DNA sequence did not correspond to mutations known to abolish the binding of E2F, it was concluded that the sequences identified corresponded to functional E2F-binding sequences.
• E2F protein In order to assess the possible functionality of WDVl, its interaction with an E2F protein was tested using a genetic screen in yeast (the so-called “one-hybrid system") which consists of placing the sequence to be studied in the promoter region of two yeast reporter genes, whose expression becomes dependent on the interaction between the putative target sequence in the promoter and the DNA binding domain of the transcription factor that recognizes it.
• This DNA binding domain can be supplied as a clone of known sequence, fused to the GAL4 activation domain.
• the cDNA coding human E2F-1 was used.
• E2F proteins normally act as dimers with members of the DP family, they are also known to be recognisable on their own, albeit with a lower affinity. Thus it was anticipated that the GAL4/E2F-1 hybrid protein alone could bind to, and activate, the chimaeric promoters.
• the experimental details were as follows: The Matchmaker One-Hybrid SystemTM (CLONTECH) was used for testing the binding properties of the WDV putative E2F binding site, according to the manufacturer's protocols.
• the first step consisted in constructing the yeast reporter strain. A double- stranded oligonucleotide with the sequence 5'-
• AATTC(TTTTGGCGGGAGAA) 3 G-3' corresponding to a tandem trimer of the WDVl putative binding site was first cloned into EcoRI - digested BluescriptSK * to make pSClHlO. The insertion was sequenced to determine the orientation of the polylinker, and promoter region of the lacZ reporter gene in pLacZi, or with
• Fusion of the human E2F1 coding sequence to the GAL4 activation domain was carried out as follows.
• a Xhol linker (5'- AATTCCGCTCGAGCG-3' ) was first inserted between the EcoRI and BamHl sites in the polylinker region of plasmid pGAD424, which contains the GAL4 activation domain (AD) ; to construct pGAD424XhoI.
• This cDNA was excised from pCMV-E2F (Kaelin W.G., Jr. Dana Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA) .
• the plasmid was first restricted at the 3 'end of the cDNA with EcoRI , end blunted with T4 DNA polymerase, and then cut on the 5' side with BamHI .
• This fragment containing the full human E2F-1 cDNA was ligated into pGAD424XhoI previously restricted with Sail, made blunt, then restricted with BamHI .
• the DNA sequence at the border between Gal4AD and E2F-1 is therefore : GAA TTC CGC TCG AGC GGA TTC CAT ATG GCC.
• the resulting plasmid is pGAD424XhoIE2F-l .
• Transformation of the reporter yeast with pGAD424XhoIE2F-l and screening of the yeast transformants was carried out as described in the manufacturer's instructions.
• the YSCilO reporter yeast strain was therefore constructed, and it contains the HIS3 and lacZ genes placed under the control of the potential E2F binding site, a tandem trimer of WDVl ( Figure 7A) .
• the tandem repeats are in the same orientation as transcription in lacZ, but not in HIS3 , which shows the opposite orientation. This should not have a negative influence on E2F activity (and in fact both reporter genes showed E2F-dependent activation, see below) since several E2F-dependent promoters, such as the DHFR promoter, have one or more E2F binding sites in an inverted orientation (Blake et al . , 1989) .
• YSCilO was transformed with a plasmid expressing the GAL4/E2F-1 hybrid protein (pGAD424XhoIE2F-l) , and tested for its ability to grow on medium lacking histidine.
• the transformed strain was a histidine auxotroph ( Figure 7B) , which was an indication of a positive interaction between E2F-1 DNA binding domain and the WDVl sequence. The interaction was further tested by monitoring in a filter assay the activity of the second reporter gene, lacZ, whose expression also turned out to be induced (not shown) .
• pYSCilO was independently transformed with pGAD424XhoI, which is the plasmid coding for the GAL4 DNA activation domain but lacking the E2F-1 binding domain. This "empty" plasmid did not give a positive interaction, discarding the possibility of spurious GAL4 binding to WDVl ( Figure 7B) .
• This hybrid protein also tested positive in a two hybrid system for interaction with the the maize RB protein ( Figure 7C) . , carried out as follows: The two hybrids assay was performed as previously described (Collin et al . , 1996) . Plasmids containing fusions of the GAL4 activation domain to the WDV Cl (pACTCl:C2iNde) (Collin et al .
• yeast strain and the transformation procedure were as described by Durfee et al . (1993) . Screening of the yeast transformants was carried out as described in the manufacturer's instructions (Matchmaker library Protocol, kit PT1020-1, CLONTECH, USA) .
• Example 5 Wheat and maize nuclear proteins that bind WDVl
• the DNA fragment used in gel retardation assays was recovered from pSClHIO, digested with EcoRI and end-labelled by filling-in with [ ⁇ 32 P] dATP and ⁇ 29 DNA polymerase 3' -5' Exo " (Bernad et al . , 1989) .
• the labelling reaction was conducted as described by Esteban et al . (1991) .
• Gel retardation assays were performed essentially as described by Frieds and Crothers (1981) .
• DNA used as non-specific competitor were synthetic oligonucleotides representing concatemers of the ACCGGGCCGG box that have been seen to bind specific proteins from maize extracts (Fenoll et al . , 1990) . They were added to the binding mix prior to the nuclear protein extract .
• GV LIRs ( Figure 8) , at positions consistent with transcriptional regulatory roles.
• the presence of sites for putative E2F-mediated regulation is in agreement with the finding that many monopartite geminiviruses encode proteins with the consensus LeuXCysXGlu RB- binding motif (Collin et al . , 1996), and that at least one bipartite geminivirus has been shown to encode a protein, AL1, " with a confirmed interaction with RB (Ach et al . , 1997) .
• Example 6 Use of transient expression in maize to test regulation of GV V-sense promoters in plants in presence and absence of effectors
• WDV has two E2F-binding sites in its bidirectional promoter, and at least one of them (WDVl) can bind the human E2F-1 protein in a genetic test (yeast one-hybrid system) , and can also bind nuclear proteins of plant origin (maize and wheat nuclear proteins) in vitro as detected by EMSAs , in a very specific way.
• WDVl E2F-binding sites in its bidirectional promoter
• WDVl E2F-binding sites in its bidirectional promoter, and at least one of them (WDVl) can bind the human E2F-1 protein in a genetic test (yeast one-hybrid system) , and can also bind nuclear proteins of plant origin (maize and wheat nuclear proteins) in vitro as detected by EMSAs , in a very specific way.
• Other GVs such as MSV, also have potential sites for binding of E2F-like proteins, and both maize and wheat cells have nuclear proteins which recognized WDVl in a specific manner
• Maize seeds were germinated for 24 hours in the dark, over wet paper filter and under sterile conditions. Seedlings were subsequently grown for 4-6 days in a growth chamber under a 16 hours photoperiod, at 24 C. Leaves were collected and laid in a Petri dish with nutrient medium (MS, 3%sucrose 0.6 %agar) for bombardment .
• MS nutrient medium
• Plasmids were prepared as follows:
• the C2 STOP codon was then mutagenized into an Sphl site using the following oligonucleotide 5' -CCG CGC TAG GAC AGC ATG CTG CGA AGC AGT G-3 ' .
• a double-stranded oligonucleotide (coding strand: 5' - TAC CCA TAC GAC GTC CCA GAC TAC GCC TGA-3') was then inserted into the blunt-ended Sphl site of pStagel to make pMUTA, in which the C2 coding sequence has therefore been modified by addition of 9 extra amino acids at its C terminus .
• pStage II The wide-type Notl/Hindlll fragment in pStage II was then replaced by the Notl/Hindlll mutagenized WDV sequence isolated from pMUTA, to make pStagelllA.
• the Cl coding sequence was isolated as an Ncol/Ndel fragment (the coordinates in the WDV genomic sequences are 2590 and 1755, respectively) after the Ndel site was made blunt using T4 DNA polymerase. It was cloned into pJIT163BgIII (Creissen et al (1995) Plant J.8, 167-178) previously restricted with Ncol and Smal to make pJIT163Cl (see Figure 11C) . As a result of the cloning strategy, the Ndel site in the WDV sequence, as well as the Smal site in pJIT163BgIII , are destroyed.
• E2F cDNA was excised from pCMV-E2F (Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, USA - also as described in Cell 70:351-364, 1992, with the exception that the PCR mutation at residue 434 has been eliminated) and used to create pGAD424Xhol E2F-1, as described above.
• E2F cDNA was EcoRI and Hindlll digested from pGAD424XhoI E2F-1 and was ligated to pJIT163 previously EcoRI linearized. This originated a ligation product fusing the 35S promoter to the E2F cDNA. The molecule was made blunt, and both blunt ends were ligated to each other to obtain pJIT163E2F, carrying the E2F cDNA under control of a double 35S promoter. DNA was coprecipitated with 1 mm diameter gold particles. Bombardments were made in a PDSIOO/He (Biorad) apparatus, at 1100 Psi .
• the activity in the presence of Cl may be due to an ability to increase plant E2F-like activity through binding to RB.
• Expression in the absence of added E2F and Cl, which may be due to promoter elements independent of the E2F sites, can be reduced by promoter deletions or other mutations. Modified promoters are tested for specificity as described above.
• pJIT39 was introduced into tomato (a dicot) . Although, under the conditions used, the basal level of expression was higher than in maize, activation by Cl could still be detected. By comparison, pBI221 was not activated by Cl .
• One component may be a GV V-sense promoter (native or modified version) fused to the DNA sequence selected to confer nematode resistance (such as dianthin or anti-HMGl, or others) - this is the nematode- resistance construct ;
• the second component is a fusion of the native GV promoter or other NFS inducible promoter, the expression pattern of which only overlaps with the GV promoter expression at NFS. This is fused to E2F-1 or to Cl (called trans - activating construct) .
• Both components may be introduced into a plant host (e.g. Arabidopsis) via Agrobacterium, and nematode resistance is tested as described hereinbefore.
• trans -activating plasmids The WDV Cl and the E2F-1 sequences in plasmids pJIT163Cl and pJIT163E2F are fused by conventional techniques to the WDV intergenic region present in pJIT39. Both plasmids may be tested in the Arabidopsis TRANSGENIC REPORTER LINES constructed in Example 3, by microinjection in NFSs, to determine their effect as trans activators of GUS.
• trans activating gene fusions may be cloned into a binary vector and used to produce transgenic arabidopsis lines expressing the trans- activators under the control of the WDV V-sense promoter.
• These TRANSGENIC ACTIVATOR LINES will be closely examined to rule out deleterious effects of Cl or E2F-1 expression outside the NFSs. If the plants are normal and fertile, they may be crossed with TRANSGENIC REPORTER LINES to determine if trans-activation leads to undesired GUS expression outside the NFSs.
• TRANSGENIC ACTIVATOR LINES may be crossed with the PRIMARY RESISTANT PLANTS produced in Example 3. The resulting progeny will be screened for normal development plus enhanced nematode resistance.
• step 2 show deleterious effects of the activators expressed under the WDV V-sense promoter, then alternative promoters also active at NFS, but with an expression pattern which only overlaps with GV V-sense at NFSs may be used (see Fenoll et al . , 1997; Barthels et al . , 1997).
• pG208dgc contains a full length MSV LIR region as in pG208, but the GC boxes previously described (Fenoll et al . , 1990) as essential for virion sense expression have been deleted.
• dissected promoters may have a lower basal expression in plant cells than the full length LIR, while maintaining responsiveness to specific activating factors, such as the Cl protein and other factors which may be associated with NFSs.
• the GC box deletion was prepared by PCR using the following primers :
• oligus GATTTCACGGGTTGGGGTTT ml3reverse : CAGGAAACAGCTATGAC
• oligonucleotides which hybridise with the sequences flanking the GC boxes, plus an additional tail with the EcoRI and two nucleotides to allow cloning were termed msv and msv loop .
• oligus an oligonucleotide previously designed in this laboratory that hybridise with GUS coding sequence, and m!3reverse , a commercial oligo (Boehringer) that binds sequences in pUC19 flanking the 5' end of the MSV LIR. EcoRI sites are underlined.
• Figure 13A shows the MSV LIR in pGUS208 and flanking regions in pG208. The positions where the oligonucleotides bind are shown by arrows. Restriction sites important for the cloning are also marked. Bellow we show the PCR products A and B.
• PCR A oligonucleotides msv and m!3reverse
• PCR B another reaction
• the PCR A product was digested with Hindlll/ EcoRI
• the PCR B product with Ncol/EcoRI
• the two fragments cloned at the same time into a backbone obtained by digesting pGUS208 with Hindlll/Ncol .
• Positive clones were confirmed by sequencing the mutated fragment, and one of them was chosen as pG208dgc.
• DNA from the plasmid was obtained and used in bombardment experiments as described in Example 6.
• pG208dgc on its own has a very low activity, around 0.28 fold the activity of pG208.
• the deleted construct still responded to the Cl protein from WDV, showing an increase over basal expression of 2.75x, similar to the increase experienced by the wild type promoter in pG208.
• Cl interacts in yeast with RB, and may activate V-sense GV promoters by binding to cellular Rb and making it release transcription factors needed for V-sense promoter activation.
• Rb-binding motif of Cl LICHE
• the oligonucleotides used are shown below.
• the mutations that change E to K in oligos OM2 and OM3 are marked in low case
• Figure 15 shows the Cl coding region with the changed amino acid, the positions of the 4 oligonucleotides used to introduce the mutation, and restriction sites important for the cloning.
• PCR1 was prepared with 0M4 and OM3 , and PCR2 with OM1 and OM2 using as a template a plasmid containing the Cl sequence, pSTAGEIIIA (see Figure 11 A) .
• a further PCR was carried out using as DNA template the combined products of PCR1 and 2, and the primers OM1 and OM4 . From this reaction a sequence representing a fragment of the Cl protein with a mutation that changes the- codon GAG (that codes for E) , to codon AAG (that codes for K) was obtained.

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