NZ545777A - Use of thionin proteins and encoding nucleic acid fragments for the modification of plant defense, disease/pest resistance and protein storage - Google Patents

Abstract

Disclosed is a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a thionin (TH) polypeptide from a ryegrass (Lolium) species, or a functionally active fragment thereof. Also disclosed are complement and antisense sequences of the above. Sequences may be used to modify disease, pest resistance, plant defense and protein storage by way of introduction to the plant via vectors, or may be used as molecular marker

Description

005014324 1 545777 MODIFICATION OF PLANT DEFENCE, DISEASE/PEST RESISTANCE AND PROTEIN STORAGE (2) This application is a divisional of New Zealand patent application 529212, the entire disclosure of which is incorporated herein by reference.

This application is the parent of a divisional application entitled "Modification of plant defence, disease/pest resistance and protein storage (3)".

The present invention relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for thionins (TH), thaumatin-like (TL) proteins, elicitor-responsive (ER) proteins and defensins (DEF) in plants and the use thereof for, inter 10 alia, modification of plant defense, disease and/or pest resistance, and/or protein ~ storage, in plants.

Thionins are a group of low-molecular-weight generally basic polypeptides, with 6 or 8 conserved cysteine residues and an L-shaped three- dimensional 10 amphipathic structure, which show a broad range of toxic effects exerted at the level of cell 15 membranes of bacteria, fungi, and higher organisms. They accumulate in plant seeds and leaves, where they may function as storage proteins, general-purpose defense toxins and contribute to the plant's defense mechanism.

Thaumatin-like proteins are acidic apoplastic or basic vacuolar proteins that show enzyme (protease, amylase) inhibitory activity, and may have defensive role against 20 pests and pathogens. Their accumulation may be induced by viral infection or correlated with osmotic adaptation.

Elicitor-responsive proteins are polypeptides from genes induced and/or activated by elicitors and/or pathogens. ER are involved in the defense response of plants to counter attack by pathogens and may deter pests and herbivores.

Defensins are small cysteine-rich peptides with antimicrobial activity produced by plants. They show in vitro antifungal properties and may contribute to plant defense.

While nucleic acid sequences encoding some TH, TL, ER and DEF have been isolated for certain species of plants, there remains a need for materials useful in modifying plant defense response, disease and/or pest resistance, and/or protein 30 storage, in a wide range of plants, particularly in forage and turf grasses and legumes, including ryegrasses, fescues and clovers, and for methods INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 ? AUG 2007 i MMAVBinrrn 2 for their use.

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

In one aspect, the present invention provides substantially purified or 5 isolated nucleic acids or nucleic acid fragments encoding TH, TL, ER and DEF from a ryegrass (Lolium) or fescue (Festuca) species and functionally active fragments and variants thereof.

The present invention also provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for a class of 10 proteins which are related to TH, TL, ER and DEF and functionally active fragments and variants thereof. Such proteins are referred to herein as TH-like, TL-like, ER-like and DEF-like, respectively. the individual or simultaneous enhancement or otherwise manipulation of TH, TL, ER and/or DEF or like gene activities in plants may enhance or otherwise 15 alter plant defense, for example plant defense against microbial attack; may enhance or otherwise alter plant disease and/or pest resistance, for example the resistance status of plants to attack by pests such as insects or nematodes; may reduce wounds or other damage caused by plant diseases and/or pests; may reduce the spread of diseases and/or pests, including pathogens and other 20 opportunistic microbes; may activate cellular responses in the plant to reduce disease, pest/and or pathogen spread; or may alter protein storage and plant developmental processes.

The individual or simultaneous enhancement or otherwise manipulation of TH, TL, ER and/or DEF or like gene activities in plants has significant 25 consequences for a range of applications in, for example, plant production and plant protection. For example, it has applications in increasing plant resistance to diseases such as fungal, bacterial and viral diseases; in increasing plant resistance to attack by pests such as insects; in increasing plant resistance to infection by pests such as nematodes; in increasing spectrum of disease 3 resistance to a wide range of diseases and/or pests including pathogens; in reducing plant damage caused by eg. herbivore predators; in reducing plant wounding caused by eg. herbivore predators; in reducing entry points for and spread of diseases and/or pests including pathogens; and in reducing the reliance 5 on chemical applications eg. fungicides, insecticides, etc. for disease and/or pest control.

Methods for the manipulation of TH, TL, ER and/or DEF or like gene activities in plants, including grass species such as ryegrasses (Lolium species) and fescues (Festuca species), and legumes such as clovers (Trifolium species) 10 may facilitate the production of, for example, pasture and turf grasses and pasture legumes with enhanced resistance to diseases and/or pests, for example insects and/or nematodes, modified plant defense response, and/or modified protein storage.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable 15 type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass (L. perenne). Perennial ryegrass (Lolium perenne L.) is a key pasture grass in temperate climates throughout the world. Perennial ryegrass is also an important turf grass.

The nucleic acid or nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, and combinations thereof.

The term "isolated" means that the material is removed from its original 25 environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or nucleic acid fragment present in a living plant is not isolated, but the same nucleic acid or nucleic acid fragment separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids or nucleic acid fragments could be part of a vector and/or such 30 nucleic acid fragments could be part of a composition, and still be isolated in that 005014324 4 such a vector or composition is not part of its natural environment.

Such nucleic acids or nucleic acid fragments could be assembled to form a consensus contig. As used herein, the term "consensus contig" refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that 5 share common or overlapping regions of sequence homology. For example, the nucleotide sequence of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acids or nucleic acid fragments, the sequences (and thus their corresponding nucleic acids or nucleic acid 10 fragments) can be assembled into a single contiguous nucleotide sequence.

Preferably, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a thionin (TH) polypeptide from a Lolium species, or a functionally active fragment or variant thereof.

Preferably, the Lolium species is Lolium perenne or Lolium arundinaceum.

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a TH or TH-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1, 3, 4, 6, 7, 9 and 35 hereto (Sequence ID Nos: 1, 3 to 19, > 20, 22 to 24, 25, 27 and 28, and 66, respectively); (b) complements of the sequences 20 recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a TL or TL-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences 25 shown in Figures 10, 12, 13, 15 and 42 hereto (Sequence ID Nos: 29, 31 to 34, 35, 37 and 38, and 68, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 AUG 2007 RFHFIX/Fn 005014324 In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an ER or ER-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 16,18 and 49 hereto (Sequence ID Nos: 39, 41 and 42, 5 and 70, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a DEF or DEF-like 10 protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 19, 21, 22, 24, 25, 27, 29, 31, 32, 56 and 62 hereto (Sequence ID Nos: 43, 45 to 51, 52, 54 and 55, 56, 58, 60, 62 and 63, 64, 72, and 74, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and 15 variants of the sequences recited in (a), (b) and (c).

By "functionally active" in relation to nucleic acids it is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of modifying plant defense response, pest and/or disease resistance, and/or protein storage, in a plant. Such variants include naturally occurring allelic variants and non-naturally occurring 20 variants. Additions, deletions, substitutions and derivatizations of one or more of the ' nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence, more preferably at least approximately 90% identity, most 25 preferably at least approximately 95% identity. Such functionally active variants and fragments include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 10 nucleotides, more preferably at least 15 nucleotides, most preferably at least 20 nucleotides.

In a further preferred embodiment the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a TH protein or INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 ? AUG 2007 DCrciwcn 5A complementary or antisense to a sequence encoding a TH protein and including a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1, 3, 4, 6, 7, 9 and 35 hereto (Sequence ID Nos: 1, 3 to 19, 20, 22 to 24, 25, 27 and 28, and 66, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

Preferably, the functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequence recited in (a), (b) or (c), respectively, and have a size of at least 20 nucleotides.

Nucleic acids or nucleic acid fragments encoding at least a portion of INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 AUG 2007 RECEIVED WO 02/088359 PCT/AU02/00539 6 several TH, TL, ER and DEF have been isolated and identified. The nucleic acids or nucleic acid fragments of the present invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols, such as 5 methods of nucleic acid hybridisation, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g. polymerase chain reaction, ligase chain reaction), is well known in the art.

For example, genes encoding other TH or TH-like, TL or TL-like, ER or ER-like and DEF or DEF-like proteins, either as cDNAs or genomic DNAs, may be 10 isolated directly by using all or a portion of the nucleic acids or nucleic acid fragments of the present invention as hybridisation probes to screen libraries from the desired plant employing the methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention may be designed and synthesized by methods known in the art. 15 Moreover, the entire sequences may be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labelling, nick translation, or end-labelling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers may be designed and used to amplify a part or all of the sequences of the present invention. The resulting 20 amplification products may be labelled directly during amplification reactions or labelled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, short segments of the nucleic acids or nucleic acid fragments of the present invention may be used in amplification protocols to amplify longer 25 nucleic acid fragments encoding homologous genes from DNA or RNA. For example, polymerase chain reaction may be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acid sequences of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' 30 end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol [Frohman et al. 7 (1988), Proc. Natl. Acad. Sci. USA 85:8998, the entire disclosure of which is incorporated herein by reference] to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Using commercially available 3' RACE and 5' RACE systems (BRL), specific 3' or 5 5' cDNA fragments may be isolated [Ohara et al. (1989), Proc. Natl. Acad. Sci. USA 86:5673; Loh et al. (1989), Science 243:217; the entire disclosures of which are incorporated herein by reference]. Products generated by the 3' and 5' RACE procedures may be combined to generate full-length cDNAs.

In a second aspect of the present invention there is provided a substantially 10 purified or isolated polypeptide from a ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of TH and TH-like, TL and TL-like, ER and ER-like, and DEF and DEF-like proteins; and functionally active fragments and variants thereof.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable 15 type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass (L. perenne).

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated TH or TH-like polypeptide includes an amino acid sequence 20 selected from the group consisting of sequences shown in Figures 2, 5, 8 and 36 hereto (Sequence ID Nos: 2, 21, 26 and 67, respectively) and functionally active fragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated TL or TL-like polypeptide includes an amino acid 25 sequence selected from the group consisting of sequences shown in Figures 11, 14 and 43 hereto (Sequence ID Nos: 30, 36 and 69, respectively) and functionally active fragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated ER or ER-like polypeptide includes an amino acid 005014324 8 sequence selected from the group consisting of sequences shown in Figures 17 and 50 hereto (Sequence ID Nos: 40 and 71, respectively) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the 5 substantially purified or isolated DEF or DEF-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 20, 23, 26, 28, 30, 33, 57 and 63 hereto (Sequence ID Nos: 44, 53, 57, 59, 61, 65, 73 and 75, respectively) and functionally active fragments and variants thereof.

By "functionally active" in relation to polypeptides it is meant that the fragment or • l0 variant has one or more of the biological properties of the proteins TH, TH-like, TL, TL-like ER, ER-like, DEF and DEF-like, respectively. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 60% 15 identity to the relevant part of the above mentioned sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 10 amino acids, 20 more preferably at least 15 amino acids, most preferably at least 20 amino acids.

In a preferred embodiment, the present invention provides a substantially purified or isolated TH polypeptide from a Lolium species, or a and functionally active fragment or variant thereof.

Preferably, the Lolium species is Lolium perenne or Lolium arundinaceum.

In a preferred embodiment, the present invention provides a substantially purified or isolated TH polypeptide including an amino acid sequence selected from the group consisting of sequences shown in Figures 2, 5, 8 and 36 hereto (Sequence ID Nos: 2, 21, 26 and 67, respectively); and functionally active fragments and variants thereof.

INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 AUG 2007 RECEIVED 8A Preferably, the functionally active fragments and variants have at least approximately 95% identity with the relevant part of the recited sequences and have a size of at least 28 amino acids.

In a further embodiment of this aspect of the invention, there is provided a polypeptide recombinantly produced from a nucleic acid or nucleic acid fragment according to the present invention. Techniques for recombinantly producing polypeptides are well known to those skilled in the art.

Availability of the nucleotide sequences of the present invention and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides may be used to immunise animals to produce polyclonal or monoclonal antibodies with specificity for INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 AUG 2007 RECEIVED 9 peptides and/or proteins including the amino acid sequences. These antibodies may be then used to screen cDNA expression libraries to isolate full-length cDNA clones of interest.

A genotype is the genetic constitution of an individual or group. Variations 5 in genotype are important in commercial breeding programs, in determining parentage, in diagnostics and fingerprinting, and the like. Genotypes can be readily described in terms of genetic markers. A genetic marker identifies a specific region or locus in the genome. The more genetic markers, the finer defined is the genotype. A genetic marker becomes particularly useful when it is 10 allelic between organisms because it then may serve to unambiguously identify an individual. Furthermore, a genetic marker becomes particularly useful when it is based on nucleic acid sequence information that can unambiguously establish a genotype of an individual and when the function encoded by such nucleic acid is known and is associated with a specific trait. Such nucleic acids and/or nucleotide 15 sequence information including single nucleotide polymorphisms (SNPs), variations in single nucleotides between allelic forms of such nucleotide sequence, can be used as perfect markers or candidate genes for the given trait.

Applicants have identified a number of SNPs of the nucleic acids or nucleic acid fragments of the present invention. These are indicated (marked with grey on 20 the black background) in the figures that show multiple alignments of nucleotide sequences of nucleic acid fragments contributing to consensus contig sequences. See for example, Figures 3, 6,12 and 21 hereto (Sequence ID Nos: 3 to 19, 22 to 24,31 to 34, and 45 to 51, respectively).

Accordingly, in a further aspect of the present invention, there is provided a 25 substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism (SNP) from a nucleic acid or nucleic acid fragment according to the present invention, or complements or sequences antisense thereto, and functionally active fragments and variants thereof.

In a still further aspect of the present invention there is provided a method 30 of isolating a nucleic acid or nucleic acid fragment of the present invention including a SNP, said method including sequencing nucleic acid fragments from a nucleic acid library.

The nucleic acid library may be of any suitable type and is preferably a cDNA library.

The nucleic acid or nucleic acid fragment may be isolated from a recombinant plasmid or may be amplified, for example using polymerase chain reaction.

The sequencing may be performed by techniques known to those skilled in the art.

In a still further aspect of the present invention, there is provided use of the nucleic acids or nucleic acid fragments of the present invention including SNPs, and/or nucleotide sequence information thereof, as molecular genetic markers.

In a still further aspect of the present invention there is provided use of a nucleic acid or nucleic acid fragment of the present invention, and/or nucleotide 15 sequence information thereof, as a molecular genetic marker.

More particularly, nucleic acids or nucleic acid fragments according to the present invention optionally including SNPs and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and in marker assisted 20 selection, particularly in ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention optionally including SNPs and/or nucleotide sequence information thereof may be used as molecular genetic markers in forage and turf grass improvement in relation to, for example, plant defense response, disease and/or pest resistance e.g. tagging 25 QTLs for resistance to fungal and bacterial pathogens, virus resistance, insect resistance, and nematode resistance. Even more particularly, sequence information revealing SNPs in allelic variants of the nucleic acids or nucleic acid fragments of the present invention and/or nucleotide sequence information thereof 11 may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in ryegrasses and fescues.

In a still further aspect of the present invention there is provided a construct including a nucleic acid or nucleic acid fragment according to the present 5 invention.

The term "construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest, in general a construct may include the gene or genes of interest, a marker gene which in some cases can also be the gene of interest and appropriate regulatory sequences. It 10 should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

In a still further aspect of the invention there is provided a vector including a 15 nucleic acid or nucleic acid fragment according to the present invention.

The term "vector" as used herein includes both cloning and expression vectors. Vectors are often recombinant molecules including nucleic acid molecules from several sources.

In a preferred embodiment of this aspect of the invention, the vector may 20 include a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment according to the present invention and a terminator; said regulatory element, nucleic acid or nucleic acid fragment and terminator being operatively linked.

By "operatively linked" is meant that said regulatory element is capable of 25 causing expression of said nucleic acid or nucleic acid fragment in a plant cell and said terminator is capable of terminating expression of said nucleic acid or nucleic acid fragment in a plant cell. Preferably, said regulatory element is upstream of said nucleic acid or nucleic acid fragment and said terminator is downstream of 12 said nucleic acid or nucleic acid fragment.

The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant 5 viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmid from Agrobacterium rhizogenes\ phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is 10 replicable, integrative or viable in the plant cell.

The regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.

Preferably the regulatory element is a promoter. A variety of promoters 15 which may be employed in the vectors of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed (eg. monocotyledon or dicotyledon). Particularly suitable constitutive promoters include 20 the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, and the rice Actin promoter.

A variety of terminators which may be employed in the vectors of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. 25 Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos), the octopine synthase (ocs) and the rbcS genes.

The vector, in addition to the regulatory element, the nucleic acid or nucleic acid fragment of the present invention and the terminator, may include further 13 elements necessary for expression of the nucleic acid or nucleic acid fragment, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker 5 genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene and the gentamycin acetyltransferase (aacC1)], and reporter genes [such as beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include 10 appropriate sequences for amplifying expression.

As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, 15 histochemical GUS assays, northern and Western blot hybridisation analyses.

Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such 20 techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.

The constructs and vectors of the present invention may be incorporated into a variety of plants, including monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and 25 turf grasses, corn, oat, sugarcane, wheat and barley), dicotyledons (such as Arabidopsis, tobacco, white clover, red clover, subterranean clover, alfalfa, eucalyptus, potato, sugar beet, canola, soybean, chickpea) and gymnosperms. In a preferred embodiment, the constructs and vectors may be used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) 30 and fescues (Festuca species), even more preferably perennial ryegrass, including forage- and turf-type cultivars. In an alternate preferred embodiment, the 14 constructs and vectors may be used to transform dicotyledons, preferably forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and lucerne (Medicago 5 sativa). Clovers, lucerne and medics are key pasture legumes in temperate climates throughout the world.

Techniques for incorporating the constructs and vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include 10 Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection. into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.

Cells incorporating the constructs and vectors of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or 20 asexually, using methods well known in the art, to produce successive generations of transformed plants.

In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a construct, vector, nucleic acid or nucleic acid fragment of the present invention.

The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the plant cell, plant, plant seed or other plant part may be from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably perennial 30 ryegrass, including both forage- and turf-type cultivars. In an alternate preferred embodiment the plant cell, plant, plant seed or other plant part may be from a dicotyledon, preferably forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and 5 lucerne (Medicago sativa).

The present invention also provides a plant, plant seed or other plant part derived from a plant cell of the present invention.

The present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.

In a further aspect of the present invention there is provided a method of modifying disease and/or pest resistance, plant defense response and/or protein storage in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or a vector according to the present invention.

By "an effective amount" it is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a 20 suitable amount and method of administration. See, for example, Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.

Using the methods and materials of the present invention, disease and/or 25 pest resistance, plant defense response and/or protein storage may be increased, decreased or otherwise modified relative to an untransformed control plant. For example, resistance to fungal diseases or resistance to bacterial disease, or plant host response to pathogen challenge, or resistance to insect pests, or resistance to nematodes may be increased or otherwise altered. Pest and/or disease 16 resistance, plant defense response and/or protein storage may be increased or otherwise modified, for example, by incorporating additional copies of a sense nucleic acid or nucleic acid fragment of the present invention. They may be decreased or otherwise modified, for example, by incorporating an antisense 5 nucleic acid or nucleic acid fragment of the present invention.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

In the Figures Figure 1 shows the consensus contig nucleotide sequence of LpTHa (Sequence ID No: 1).

Figure 2 shows the deduced amino acid sequence of LpTHa (Sequence ID No: 2).

Figure 3 shows the nucleotide sequences of the nucleic acid fragments 15 contributing to the consensus contig sequence LpTHa (Sequence ID Nos: 3 to 19).

Figure 4 shows the consensus contig nucleotide sequence of LpTHb (Sequence ID No: 20).

Figure 5 shows the deduced amino acid sequence of LpTHb (Sequence ID No: 20 21).

Figure 6 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpTHb (Sequence ID Nos: 22 to 24).

Figure 7 shows the consensus contig nucleotide sequence of LpTHc (Sequence 25 ID No: 25). 17 Figure 8 shows the deduced amino acid sequence of LpTHc (Sequence ID No: 26).

Figure 9 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpTHc (Sequence ID Nos: 27 and 5 28).

Figure 10 shows the consensus contig nucleotide sequence of LpTLa (Sequence ID No: 29).

Figure 11 shows the deduced amino acid sequence of LpTLa (Sequence ID No: 30).

Figure 12 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpTLa (Sequence ID Nos: 31 to 34).

Figure 13 shows the consensus contig nucleotide sequence of LpTLb (Sequence ID No: 35).

Figure 14 shows the deduced amino acid sequence of LpTLb (Sequence ID No: 36).

Figure 15 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpTLb (Sequence ID Nos: 37 and 38).

Figure 16 shows the consensus contig nucleotide sequence of LpERa (Sequence ID No: 39).

Figure 17 shows the deduced amino acid sequence of LpERa (Sequence ID No: 40).

Figure 18 shows the nucleotide sequences of the nucleic acid fragments 25 contributing to the consensus contig sequence LpERa (Sequence ID Nos: 41 and 18 42).

Figure 19 shows the consensus contig nucleotide sequence of LpDEFa (Sequence ID No: 43).

Figure 20 shows the deduced amino acid sequence of LpDEFa (Sequence ID No: 5 44).

Figure 21 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpDEFa (Sequence ID Nos: 45 to 51).

Figure 22 shows the consensus contig nucleotide sequence of LpDEFb 10 (Sequence ID No: 52).

Figure 23 shows the deduced amino acid sequence of LpDEFb (Sequence ID No: 53).

Figure 24 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpDEFb (Sequence ID Nos: 54 15 and 55).

Figure 25 shows the nucleotide sequence of LpDEFc (Sequence ID No: 56).

Figure 26 shows the deduced amino acid sequence of LpDEFc (Sequence ID No: 57).

Figure 27 shows the nucleotide sequence of LpDEFd (Sequence ID No: 58).

Figure 28 shows the deduced amino acid sequence of LpDEFd (Sequence ID No: 59).

Figure 29 shows the consensus contig nucleotide sequence of LpDEFe (Sequence ID No: 60).

WO 02/088359 PCT/AU02/00539 19 Figure 30 shows the deduced amino acid sequence of LpDEFe (Sequence ID No: 61).

Figure 31 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpDEFe (Sequence ID Nos: 62 5 and 63).

Figure 32 shows the nucleotide sequence of LpDEFf (Sequence ID No: 64).

Figure 33 shows the deduced amino acid sequence of LpDEFf (Sequence ID No: 65).

Figure 34 shows a plasmid map of the cDNA encoding perennial ryegrass THa.

Figure 35 shows the nucleotide sequence of perennial ryegrass THa cDNA (Sequence ID No: 66).

Figure 36 shows the deduced amino acid sequence of perennial ryegrass THa cDNA (Sequence ID No: 67).

Figure 37 shows plasmid maps of sense and antisense constructs of LpTHa in 15 pDH51 transformation vector.

Figure 38 shows plasmid maps of sense and antisense constructs of LpTHa in pKYLX71:35S2 binary transformation vector.

Figure 39 shows plasmid maps of sense and antisense constructs of LpTHa in pPZP221:35S2 binary transformation vector.

Figure 40 shows screening by Southern hybridisation for RFLPs using LpTHa as a probe.

Figure 41 shows a plasmid map of the cDNA encoding perennial ryegrass TLa. Figure 42 shows the nucleotide sequence of perennial ryegrass TLa cDNA (Sequence ID No: 68).

Figure 43 shows the deduced amino acid sequence of perennial ryegrass TLa cDNA (Sequence ID No: 69).

Figure 44 shows plasmid maps of sense and antisense constructs of LpTLa in 5 pDH51 transformation vector.

Figure 45 shows plasmid maps of sense and antisense constructs of LpTLa in pKYLX71:35S2 binary transformation vector.

Figure 46 shows plasmid maps of sense and antisense constructs of LpTLa in pPZP221:35S2 binary transformation vector.

Figure 47 shows screening by Southern hybridisation for RFLPs using LpTLa as a probe.

Figure 48 shows a plasmid map of the cDNA encoding perennial ryegrass ERa.

Figure 49 shows the nucleotide sequence of perennial ryegrass ERa cDNA (Sequence ID No: 70).

Figure 50 shows the deduced amino acid sequence of perennial ryegrass ERa cDNA (Sequence ID No: 71).

Figure 51 shows plasmid maps of sense and antisense constructs of LpERa in pDH51 transformation vector.

Figure 52 shows plasmid maps of sense and antisense constructs of LpERa in 20 pKYLX71:35S2 binary transformation vector.

Figure 53 shows plasmid maps of sense and antisense constructs of LpERa in pPZP221:35S2 binary transformation vector.

Figure 54 shows screening by Southern hybridisation for RFLPs using LpERa as a WO 02/088359 PCT/AU02/00539 21 probe.

Figure 55 shows a plasmid map of the cDNA encoding perennial ryegrass DEFa.

Figure 56 shows the nucleotide sequence of perennial ryegrass DEFa cDNA (Sequence ID No: 72).

Figure 57 shows the deduced amino acid sequence of perennial ryegrass DEFa cDNA (Sequence ID No: 73).

Figure 58 shows plasmid maps of sense and antisense constructs of LpDEFa in pDH51 transformation vector.

Figure 59 shows plasmid maps of sense and antisense constructs of LpDEFa in 10 pPZP221:35S2 binary transformation vector.

Figure 60 shows screening by Southern hybridisation for RFLPs using LpDEFa as a probe.

Figure 61 shows a plasmid map of the cDNA encoding perennial ryegrass DEFe.

Figure 62 shows the nucleotide sequence of perennial ryegrass DEFe cDNA 15 (Sequence ID No: 74).

Figure 63 shows the deduced amino acid sequence of perennial ryegrass DEFe cDNA (Sequence ID No: 75).

Figure 64 shows plasmid maps of sense and antisense constructs of LpDEFe in pDH51 transformation vector.

Figure 65 shows plasmid maps of sense and antisense constructs of LpDEFe in pKYLX71:35S2 binary transformation vector.

Figure 66 shows plasmid maps of sense and antisense constructs of LpDEFe in pPZP221:35S2 binary transformation vector. 22 Figure 67 shows screening by Southern hybridisation for RFLPs using LpDEFe as a probe.

Figure 68 shows A, infiltration of Arabidopsis plants; B, selection of transgenic Arabidopsis plants on medium containing 75 /vg/ml gentamycin; C, young 5 transgenic Arabidopsis plants; D, E, two representative results of real-time PCR analysis of Arabidopsis transformed with chimeric genes involved in plant defense, disease/pest resistance and protein storage.

Figure 69 shows the genetic map detailing the relation of perennial ryegrass genes involved in plant defense, disease/pest resistance and protein storage with 10 the linkage groups in perennial ryegrass.

Figure 70 shows a subgrid of a microarray for the expression profiling of perennial ryegrass genes involved in plant defense, disease/pest resistance and protein storage.

EXAMPLE 1 Preparation of cDNA libraries, isolation and sequencing of cDNAs coding for TH, TH-like, TL, TL-like, ER, ER-like, DEF and DEF-like proteins from perennial ryegrass (Lolium perenne) cDNA libraries representing mRNAs from various organs and tissues of 20 perennial ryegrass (Lolium perenne) were prepared. The characteristics of the libraries are described below (Table 1). 23 TABLE 1 cDNA libraries from perennial ryegrass (Lolium perenne) Library Organ/Tissue 01 rg Roots from 3-4 day old light-grown seedlings 02rg Leaves from 3-4 day old light-grown seedlings 03rg Etiolated 3-4 day old dark-grown seedlings 04rg Whole etiolated seedlings (1-5 day old and 17 days old) 05rg Senescing leaves from mature plants 06rg Whole etiolated seedlings (1-5 day old and 17 days old) 07rg Roots from mature plants grown in hydroponic culture 08rg Senescent leaf tissue 09rg Whole tillers and sliced leaves (0,1,3, 6,12 and 24 h after harvesting) 10rg Embryogenic suspension-cultured cells 11 rg Non-embryogenic suspension-cultured cells 12rg Whole tillers and sliced leaves (0,1,3, 6,12 and 24 h after harvesting) 13rg Shoot apices including vegetative apical meristems 14rg Immature inflorescences including different stages of inflorescence meristem and inflorescence development 15rg Defatted pollen 16rg Leaf blades and leaf sheaths (rbcL, rbcS, cab, wir2A subtracted) 17rg Senescing leaves and tillers 18rg Drought-stressed tillers (pseudostems from plants subjected to PEG-simulated drought stress) 19rg Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with half-strength salts) (1,2, 3,4,5, 6,24 and 48 h after transfer) 20rg Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with double-strength salts) (1,2,3,4, 5, 6,24 and 48 h after transfer) 21 rg Drought-stressed tillers (pseudostems from plants subjected to PEG-simulated drought stress) 22rg Spikelets with open and maturing florets 23rg Mature roots (specific subtraction with leaf tissue) WO 02/088359 PCT/AU02/00539 24 The cDNA libraries may be prepared by any of many methods available. For example, total RNA may be isolated using the Trizol method (Gibco-BRL, USA) or the RNeasy Plant Mini kit (Qiagen, Germany), following the 5 manufacturers' instructions. cDNAs may be generated using the SMART PCR cDNA synthesis kit (Clontech, USA), cDNAs may be amplified by long distance polymerase chain reaction using the Advantage 2 PCR Enzyme system (Clontech, USA), cDNAs may be cleaned using the GeneClean spin column (Bio 101, USA), tailed and size fractionated, according to the protocol provided by Clontech. The 10 cDNAs may be introduced into the pGEM-T Easy Vector system 1 (Promega, USA) according to the protocol provided by Promega. The cDNAs in the pGEM-T Easy plasmid vector are transfected into Escherichia coli Epicurian coli XL10-Gold ultra competent cells (Stratagene, USA) according to the protocol provided by Stratagene.

Alternatively, the cDNAs may be introduced into plasmid vectors for first preparing the cDNA libraries in Uni-ZAP XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA, USA). The Uni-ZAP XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the 20 plasmid vector pBiuescript. In addition, the cDNAs may be introduced directly into precut pBiuescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into E. coli DH10B cells according to the manufacturer's protocol (GIBCO BRL Products).

Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared 25 from randomly picked bacterial colonies containing recombinant plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Plasmid DNA preparation may be performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocol provided by Qiagen. Amplified 30 insert DNAs are sequenced in dye-terminator sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"). The resulting ESTs are analysed using an Applied Biosystems ABI3700 sequence analyser. wo 02/088359 pct/au02/00539 EXAMPLE 2 DNA sequence analyses The cDNA clones encoding TH, TH-like, TL, TL-like, ER, ER-like, DEF and 5 DEF-like proteins were identified by conducting BLAST [Basic Local Alignment Search Tool; Altschul etal. (1990), J. Mol. Biol. 215:403-410] searches. The cDNA sequences obtained were analysed for similarity to all publicly available DNA sequences contained in the eBioinformatics nucleotide database using the BLASTn algorithm provided by the National Center for Biotechnology Information 10 (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the SWISS-PROT protein sequence database using BLASTx algorithm (v 2.0.1) [Gish and States (1993), Nature Genetics 3:266-272] provided by the NCBI.

The cDNA sequences obtained and identified were then used to identify 15 additional identical and/or overlapping cDNA sequences generated using the BLASTn algorithm. The identical and/or overlapping sequences were subjected to a multiple alignment using the CLUSTALw algorithm, and to generate a consensus contig sequence derived from this multiple sequence alignment. The consensus contig sequence was then used as a query for a search against the 20 SWISS-PROT protein sequence database using the BLASTx algorithm to confirm the initial identification.

EXAMPLE 3 Identification and full-length sequencing of cDNAs from perennial ryegrass 25 (Lolium perenne) encoding THa, TLa, ERa, DEFa and DEFe proteins To fully characterise for the purposes of the generation of probes for hybridisation experiments and the generation of transformation vectors, a set of perennial ryegrass cDNAs encoding THa, TLa, ERa, DEFa and DEFe proteins 26 was identified and fully sequenced.

Full-length cDNAs were identified from our EST sequence database using relevant published sequences (NCBI databank) as queries for BLAST searches. Full-length cDNAs were identified by alignment of the query and hit sequences 5 using Sequencher (Gene Codes Corp., Ann Arbor, Ml 48108, USA). The original plasmid was then used to transform chemically competent XL-1 cells (prepared in-house, CaCb protocol). After colony PCR (using HotStarTaq, Qiagen) a minimum of three PCR-positive colonies per transformation were picked for initial sequencing with M13F and M13R primers. The resulting sequences were aligned 10 with the original EST sequence using Sequencher to confirm identity and one of the three clones was picked for full-length sequencing, usually the one with the best initial sequencing result.

Sequencing was completed by primer walking, i.e. oligonucleotide primers were designed to the initial sequence and used for further sequencing. In most 15 cases the sequencing could be done from both 5' and 3' end. The sequences of the internal oligonucleotide primers are shown in Table 2. In some instances, however, an extended poly-A tail necessitated the sequencing of the cDNA to be completed from the 5' end.

Contigs were then assembled in Sequencher. The contigs include the 20 sequences of the SMART primers used to generate the initial cDNA library as well as some pGEM-T Easy vector sequence with the EcoRI cut site highlighted in bold both at the 5' and 3' end.

Plasmid maps and the full cDNA sequences of perennial ryegrass THa, TLa, ERa, DEFa and DEFe were obtained (Figures 34, 35, 41, 42, 48, 49, 55, 56, 25 61 and 62).

WO 02/088359 PCT/AU02/00539 27 TABLE 2 List of primers used for sequencing of the full-length cDNAs gene name clone ID sequencing primer primer sequence (5'->3") LpDEFa 14rg2AsA06 14rg2AsA06.f1 ttgctaatcttgtgcgcg LpDEFe 06rg2TsH06 06rg2TsH06.f1 tggcactacttgaactcg LpERa 17rg1MsF06 17rg1MsF06.f1 gaagcaaggatccctctgg Note: In most instances the sequence information provided by reactions performed with M13F and M13R was sufficient and no additional primers were needed.

EXAMPLE 4 Development of transformation vectors containing chimeric genes with THa, 10 TLa, ERa, DEFa and DEFe cDNA sequences from perennial ryegrass To alter the expression of THa, TLa, ERa, DEFa and DEFe, through antisense and/or sense suppression technology and for over-expression of these proteins in transgenic plants, a set of sense and antisense transformation vectors was produced. cDNA fragments were generated by high fidelity PCR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) contained restriction sites for EcoRI and Xbal for directional and non-directional cloning into the target vector. After PCR amplification and restriction digest with the appropriate restriction enzyme (usually Xbal), the cDNA fragments were cloned 20 into the corresponding site in pDH51, a pUC18-based transformation vector containing a CaMV 35S expression cassette. The orientation of the constructs (sense or antisense) was checked by DNA sequencing through the multi-cloning site of the vector. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs of perennial ryegrass THa, TLa, ERa, DEFa 25 and DEFe in sense and antisense orientations under the control of the CaMV 35S promoter were generated (Figures 37,44,51,58 and 64).

WO 02/088359 PCT/AU02/00539 28 TABLE 3 List of primers used to PCR-amplify the open reading frames gene name clone ID primer primer sequence (5'->3') LpDEFa 14rg2AsA06 14rg2AsA06.f gaattctagattagcgcgatccagacatccagtcc 14rg2AsA06.r gaattctagatcgatccaacggaggaactaatcgc LpDEFe 06rg2TsH06 06rg2TsH06.f gaattctagaagagctagcaccacccacctcgcc 06rg2TsH06.r gaattctagatccacgacctgctccacagctcc LpERa 17rg1MsF06 17rg1MsF06.f gaattctagacaccgggaggaccaagaagatgg 17rg1MsF06.r gaattctagaagttcagcaaagcatccacttcc LpTHa 06rg1 EsE07 06rg1 EsE07.f gaattctagaatcgagctcgagcaagtagcgcagg 06rg1EsE07.r gaattctagaatatatagcgatgcatgcatatgg LpTLa 08rg1 KsH03 08rg1KsH03.f gaattctagaacaaacaggagatcgaaacaatgg 08rg1 KsH03.r gaattctagagacttatgatgggcagaagacgacc EXAMPLES Development of binary transformation vectors containing chimeric genes with THa, TLa, ERa, DEFa and DEFe cDNA sequences from perennial ryegrass To alter the expression of the enzymes THa, TLa, ERa, DEFa and DEFe, through antisense and/or sense suppression technology and for over-expression 10 of these proteins in transgenic plants, a set of sense and antisense transformation vectors was produced. cDNA fragments were generated by high fidelity PCR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) contained restriction sites for EcoRI and Xbal for directional and non-directional cloning into 15 the target vector. After PCR amplification and restriction digest with the appropriate restriction enzyme (usually Xbal), the cDNA fragments were cloned into the corresponding site in pKYLX71:35S2, a binary transformation vector. The vector contains between the left and the right border the plant selectable marker gene nptll under the control of the nos promoter and nos terminator and an 29 expression cassette with a CaMV 35S promoter with a duplicated enhancer region and an rbcS terminator (An et al., 1985; Schardl et al., 1987). Alternatively, the PCR fragments were cloned into a modified pPZP binary vector (Hajdukiewicz et al., 1994). The pPZP221 vector was modified to contain the 35S2 cassette from 5 pKYLX71:35S2 as follows. pKYLX71:35S2 was cut with Clal. The 5' overhang was filled in using Klenow and the blunt end was A-tailed with Taq polymerase. After cutting with EcoRl, the 2kb fragment with an EcoRI-compatible and a 3'-A tail was gel-purified. pPZP221 was cut with Hindlll and the resulting 5' overhang filled in and T-tailed with Taq polymerase. The remainder of the original pPZP221 multi-10 cloning site was removed by digestion with EcoRl, and the expression cassette cloned into the EcoRl site and the 3' T overhang restoring the Hindlll site. This binary vector contains between the left and right border the plant selectable marker gene aaaC1 under the control of the 35S promoter and 35S terminator and the pKYLX71:35S2-derived expression cassette with a CaMV 35S promoter 15 with a duplicated enhancer region and an rbcS terminator.

The orientation of the constructs (sense or antisense) was checked by restriction enzyme digest. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs of perennial ryegrass THa, TLa, ERa, DEFa and DEFe in sense and antisense orientations under the control of the CaMV 20 35S2 promoter were generated (Figures 45, 46, 52, 53, 59, 65 and 66).

EXAMPLE 6 Production and analysis of transgenic Arabidopsis plants carrying chimeric perennial ryegrass genes THa, TLa, ERa, DEFa and DEFe in sense and antisense orientations involved in plant defense, disease/pest resistance 25 and protein storage A set of transgenic Arabidopsis plants carrying chimeric perennial ryegrass genes involved in plant defense, disease/pest resistance and protein storage were produced. pPZP221-based transformation vectors with LpTHa, LpTLa, LpERa, LpDEFa and LpDEFe cDNAs comprising the full open reading frame sequences in sense and antisense orientations under the control of the CaMV 35S promoter with duplicated enhancer region (35S2) were generated as detailed in Example 6.

Agrobacterium-mediated gene transfer experiments were performed using 5 these transformation vectors.

The production of transgenic Arabidopsis plants carrying chimeric perennial ryegrass genes involved in plant defense, disease/pest resistance and protein storage under the control of the CaMV 35S promoter with duplicated enhancer region (35S2) is described here in detail.

Preparation of Arabidopsis plants Seedling punnets were filled with Debco seed raising mixture (Debco Pty. Ltd.) to form a mound. The mound was covered with two layers of anti-bird netting secured with rubber bands on each side. The soil was saturated with water and enough seeds {Arabidopsis ihaliana ecotype Columbia, Lehle Seeds #WT-02) 15 sown to obtain approximately 15 plants per punnet. The seeds were then vernalised by placing the punnets at 4 SC. After 48 hours the punnets were transferred to a growth room at 22 eC under fluorescent light (constant illumination, 55 /vmolm'V1) and fed with Miracle-Gro (Scotts Australia Pty. Ltd.) once a week. Primary bolts were removed as soon as they appeared. After 4-6 20 days the secondary bolts were approximately 6 cm tall, and the plants were ready for vacuum infiltration.

Preparation of Agrobacterium Agrobacterium tumefaciens strain AGL-1 were streaked on LB medium containing 50 ^g/ml rifampicin and 50 //g/ml kanamycin and grown at 27 SC for 48 25 hours. A single colony was used to inoculate 5 ml of LB medium containing 50 //g/ml rifampicin and 50 //g/ml kanamycin and grown over night at 27 SC and 250 rpm on an orbital shaker. The overnight culture was used as an inoculum for 500 ml of LB medium containing 50 /vg/ml kanamycin only. Incubation was over night at 27 fiC and 250 rpm on an orbital shaker in a 21 Erlenmeyer flask. 31 The overnight cultures were centrifuged for 15 min at 5500 xg and the supernatant discarded. The cells were resuspended in 1 I of infiltration medium [5% (w/v) sucrose, 0.03% (v/v) Silwet-L77 (Vac-ln-Stuff, Lehle Seeds #VIS-01)J and immediately used for infiltration.

Vacuum infiltration The Agrobacterium suspension was poured into a container (Decor Tellfresh storer, #024) and the container placed inside the vacuum desiccator (Bel Art, #42020-0000). A punnet with Arabidopsis plants was inverted and dipped into the Agrobacterium suspension and a gentle vacuum (250 mm Hg) was applied for 10 2 min. After infiltration, the plants were returned to the growth room where they were kept away from direct light overnight. The next day the plants were returned to full direct light and allowed to grow until the siliques were fully developed. The plants were then allowed to dry out, the seed collected from the siliques and either stored at room temperature in a dry container or used for selection of 15 transformants.

Selection of transformants Prior to plating the seeds were sterilised as follows. Sufficient seeds for one 150 mm petri dish (approximately 40 mg or 2000 seeds) were placed in a 1.5 ml microfuge tube. 500 //I 70% ethanol were added for 2 min and replaced by 500 jj\ 20 sterilisation solution (H20:4% chlorine:5% SDS, 15:8:1). After vigorous shaking, the tube was left for 10 min after which time the sterilisation solution was replaced with 500 jj\ sterile water. The tube was shaken and spun for 5 sec to sediment the seeds. The washing step was repeated 3 times and the seeds were left covered with approximately 200 //I sterile water.

The seeds were then evenly spread on 150 mm petri dishes containing germination medium (4.61 g Murashige & Skoog salts, 10 g sucrose, 1 ml 1 M KOH, 2 g Phytagel, 0.5 g MES and 1 ml 1000x Gamborg's B-5 vitamins per litre) supplemented with 250 //g/ml timetin and 75 pg/ml gentamycin. After vernalisation for 48 hours at 4 BC the plants were grown under continuous fluorescent light (55 30 pmo\ m-2s-1) at 22 eC to the 6 - 8 leaf stage and transferred to soil. 32 Preparation of genomic DNA 3-4 leaves of Arabidopsis plants regenerated on selective medium were harvested and freeze-dried. The tissue was homogenised on a Retsch MM300 mixer mill, then centrifuged for 10 min at 1700xg to collect cell debris. Genomic 5 DNA was isolated from the supernatant using Wizard Magnetic 96 DNA Plant System kits (Promega) on a Biomek FX (Beckman Coulter). 5 //I of the sample (50 !j\) were then analysed on an agarose gel to check the yield and the quality of the genomic DNA.

Analysis of DNA using real-time PCR Genomic DNA was analysed for the presence of the transgene by real-time PCR using SYBR Green chemistry. PCR primer pairs (Table 4) were designed using MacVector (Accelrys). The forward primer was located within the 35S2 promoter region and the reverse primer within the transgene to amplify products of approximately 150 bp as recommended. The positioning of the forward primer 15 within the 35S2 promoter region guaranteed that homologous genes in Arabidopsis were not detected.

//I of each genomic DNA sample was run in a 50 //I PCR reaction including SYBR Green on an ABI (Applied Biosystems) together with samples containing DNA isolated from wild type Arabidopsis plants (negative control), 20 samples containing buffer instead of DNA (buffer control) and samples containing the plasmid used for transformation (positive plasmid control).

Plants were obtained after transformation with all chimeric constructs and selection on medium containing gentamycin. The selection process and two representative real-time PCR analyses are shown in Figure 68. 33 TABLE 4 List of primers used for Real-time PCR analysis of Arabidopsis plants transformed with chimeric perennial ryegrass genes involved in plant defense, disease/pest resistance and protein storage construct primer 1 (forward) primer 2 (reverse) LpDEFasense A.CAATCCCACTATCCTTCGCAAGAC ACCTTCGTCGTCCCCATCTCTG LpDEFaanti ATTTCATTTGGAGAGGACACGCTG CAAGTGCTTCTGCAAGAGGGTCTG LpDEFesense AATCCCACTATCCTTCGCAAGACC TTCCAGAATCCTTCCCTCATTGG LpDEFeanti ttggagaggacacgctgaaatcac TGGGTACTGGAAACAGAGCATTGC LpERasense rTGGAGAGGACACGCTGAAATCAC TGTTGCACAGGTAGTCGGTGTTCTC LpERaanti rTTCATTTGGAGAGGACACGCTG GGAGGCTTAGCATTTATTACCCGAG LpTHasense rCATTTCATTTGGAGAGGACACGC 3AGGAGCATAACCAGAACCACCG LpTHaanti AATCCCACTATCCTTCGCAAGACC CCCGTGCATGTCCAACAAGAAC LpTLasense rCATTTCATTTGGAGAGGACACGC AAGGGGCACTGGTTGGTGATGC LpTLaanti AGTTCATTTCATTTGGAGAGGACACG 3CGTACCAGCAGCCCAACG EXAMPLE 7 Genetic mapping of perennial ryegrass genes involved in plant defense, disease/pest resistance and protein storage The cDNAs representing genes involved in plant defense, disease/pest 10 resistance and protein storage were amplified by PCR from their respective plasmids, gel-purified and radio-labelled for use as probes to detect restriction fragment length polymorphisms (RFLPs). RFLPs were mapped in the Fi (first generation) population, NA6 x AU6. This population was made by crossing an individual (NA6) from a North African ecotype with an individual (AU6) from the 15 cultivar Aurora, which is derived from a Swiss ecotype. Genomic DNA of the 2 parents and 114 progeny was extracted using the 1 x CTAB method of Fulton et al. (1995).

Probes were screened for their ability to detect polymorphism using the 34 DNA (10 pig) of both parents and 5 Fi progeny restricted with the enzymes Dral, EcoRl, EcoRV or Hindlll. Hybridisations were carried out using the method of Sharp et al. (1988). Polymorphic probes were screened on a progeny set of 114 individuals restricted with the appropriate enzyme (Figures 40,47,54, 60 and 67).

RFLP bands segregating within the population were scored and the data was entered into an Excel spreadsheet. Alleles showing the expected 1:1 ratio were mapped using MAPMAKER 3.0 (Lander et al., 1987). Alleles segregating from, and unique to, each parent, were mapped separately to give two different linkage maps. Markers were grouped into linkage groups at a LOD of 5.0 and 10 ordered within each linkage group using a LOD threshold of 2.0.

Loci representing genes involved .in plant defense, disease/pest resistance and protein storage mapped to the linkage groups as indicated in Table 5 and in Figure 69. These gene locations can now be used as candidate genes for quantitative trait loci for improvement and modification of plant defense, resistance 15 to pests and diseases and storage of proteins.

TABLE 5 Map locations of ryegrass genes encoding protein involved in plant defense, disease/pest resistance and protein storage across two genetic linkage maps of perennial ryegrass Probe Polymorphic Mapped Locus Linkage with group NA6 AU6 LpDEFa Y Dra I LpDEFa 6 6 LpDEFc Y Eco RV LpDEFc LpDEFf Y Dra I LpDEFf LpERa Y Eco RV LpERa 2 2 LpTHb Y Dra I LpTHb 1 LpTHc Y Hind III LpTHc. 1 7 7 LpTHc.2 7 7 LpTLb Y Dra I LpTLb EXAMPLE 8 Expression profiling of cDNAs encoding proteins involved in plant defense, disease/pest resistance and protein storage cDNAs encoding proteins involved in plant defense, disease/pest 10 resistance and protein storage were PCR amplified and purified. The amplified products were spotted on each amino-silane coated glass slide (CMT-GAPS, Corning, USA) using a microarrayer MicroGrid (BioRobotics, UK). Spotting solution was also spotted in every subgrid of the microarray as negative and background controls.

Table 6 gives details on the tissues used to extract total RNA. Fluorescence labelled probes were synthesis by reversed transcribing RNA and incorporating Cyanine 3 or 5 labelled dCTP. The probes were hybridised onto microarrays. In 36 each case the experiment was repeated on two microarrays. After hybridisation for 16 hours (overnight), the microarrays were washed and scanned using a confocal laser scanner (ScanArray 3000, Packard, USA). The images obtained were quantified using Imagene 4.1 (BioDiscovery, USA). Data were scaled to a factor of 5 2000 across all experiments and judged as not present (-), low expression (+), medium expression (++), high expression (+++) and highest expression (++++) (Table 7).

TABLE 6 List of hybridisation probes used in expression profiling of perennial 10 ryegrass genes encoding proteins involved in plant defense, disease/pest resistance and protein storage Hybridisation probe for microarrays Organ specificity (3-months old plants Leaf blade grown hydroponically) - • Sheat Root Seed Seedling grown under light condition -day old shoot (5LS) 7-day old shoot (7LS) -day old shoot (10LS) -day old root (5LR) 7-day old root (7LR) -day old root (10LR) Seedling grown under dark condition -day old shoot (5DS) 7-day old shoot (7DS) -day old shoot (10DS) -day old root (5DR) 7-day old root (7DR) -day old root (10DR) 3 TABLE 7 2 Results of expression profiling of ryegrass genes encoding proteins Involved in plant defense, disease/pest resistance and protein storage Clone Clone id Gene id length Sheath leaf Root seed 5LS 7LS 10LS 5LR 7LR 10LR 5DS 7DS 10DS 5DR 7DR 10DR name in patent THa 06rq1 EsE07 DdrTHGl WHEAT11684 full ++ ++ -+ ++++ - + - + + + + + - - + THb 06rg2TsF02 DdrTHG_PETIN-12217 full + ++ - - ++ + + + + + +++ + - - + THc 20rq1XsD11 DdrSIAI SORBI24037 full - + • ++++ + + - + + + + + + + + TLb 06ra1TsH06 DdrTLP ORYSA-11718 full + ++ ++ ++ ++ + ++ ++ ++ ++ + + ++ ++ ++ DEFa 14rg2AsA06 DdrP322 SOLTU11598 full + +++ + + +++ + ++ + + + ++++ +++ + + + DEFb 17ra1QsF11 DdrPAFP PHYAM21417 full - - - - - - - - - - - - - - - DEFe 01rg1QsC09 NxxFSTL_FLAB110539 partial + ++++ - - - - - + ++ + - + + + + DEFd 01rg1JsF05 NppF4ST FLACH8661 partial + + + + ++ + + + + + + + ++ + + DEFe 06rg2TsH06 NppFSTL ARATH11640 full - + -t* ;+ ;+++ ;+ ;+ ;+ ;+ ;++ ;++ ;+ ;+++ ;+ ;+ ;DEFf ;06rg2MsH08 ;NppFSTL ARATH12084 ;full ;+ ;+ ;+ ;+ ;+ ;+ ;+ . ;+ ;++ ;++ ;++ ;+ ;+ ;++ ;++ ;++ ;n g ;O ;55 o v% o* \o 38 REFERENCES Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215:403-410.

An, G., Watson, B.D., Stachel, S., Gordon, M.P., Nester, E.W. (1985) New cloning 5 vehicles for transformation of higher plants. The EMBO Journal 4,227-284 Feinberg, A.P., Vogelstein, B. (1984). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.

Frohman M.A., Dush M.K., Martin G.R. (1988) Rapid production of full-length 10 cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA. 85:8998-9002.

Fulton L.L., Hillier L.D., Wilson R.K. (1995) Large-scale complementary DNA sequencing methods. Methods Cell Biol. 48:571-582.

Gish, W., States, D.J. (1993) Identification of protein coding regions by database 15 similarity search. Nature Genetics 3:266-272 Hajdukiewicz P., Svab Z., Maliga P. (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25:989-994.

Lander, E.S., Green P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E., 20 Newburg, L. (1987). MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations. Genomics 1:174-181.

Loh, E.Y., Elliott, J.F., Cwirla, S., Lanier, L.L., Davis, M.M. (1989). Polymerase chain reaction with single-sided specificity: Analysis of T-cell receptor delta 25 chain. Science 243:217-220 39 Ohara, O., Dorit, R.L., Gilbert, W. (1989). One-sided polymerase chain reaction: The amplification of cDNA. Proc. Natl. Acad. Sci. USA 86:5673-5677 Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. Coid Spring Harbour Laboratory Press Schardl, C.L., Byrd, A.D., Benzion, G.( Altschuler, M.A., Hildebrand, D.F., Hunt, A.G. (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61:1-11 Sharp, P.J., Kreis, M., Shewry, P.R., Gale, M.D. (1988). Location of a-amylase sequences in wheat and its relatives. Theor. Appl. Genet. 75:286-290.

Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

It will also be understood that the term "comprises" (or its grammatical 15 variants) as used in this specification is equivalent to the term "includes" and should not be taken as excluding the presence of other elements or features.

Documents cited in this specification are for reference purposes only and their inclusion is not an acknowledgement that they form part of the common general knowledge in the relevant art. 005014324 40

Claims (23)

1. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding a thionin (TH) polypeptide from a Lolium species, or a functionally active fragment or variant thereof. 5 2. A nucleic acid or nucleic acid fragment according to Claim 1, wherein said

Lolium species is Lolium perenne or Lolium arundinaceum.

3. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding a TH protein or complementary or antisense to a sequence encoding a TH protein and including a nucleotide sequence selected from the group consisting of (a) 10 sequences shown in Figures 1, 3, 4, 6, 7, 9 and 35 hereto (Sequence ID Nos: 1, 3 to 19, 20, 22 to 24, 25, 27 and 28, and 66, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

4. A nucleic acid or nucleic acid fragment according to Claim 3 wherein said 15 functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequence recited in (a), (b) or (c), respectively, and have a size of at least 20 nucleotides. i

5. A construct including a nucleic acid or nucleic acid fragment according to any one of Claims 1-4. 20

6. A vector including a nucleic acid or nucleic acid fragment according to any one of Claims1-4.

7. A vector according to Claim 6, further including a promoter and a terminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked. 25 8. A plant cell, plant, plant seed or other plant part, including a construct according to Claim 5 or a vector according to Claim 6 or 7.

INTELLECTUAL PROPERTY OFFICE OF N.Z. \ l AUG 2007 received 005014324 41

9. A plant, plant seed or other plant part derived from a plant cell or plant according to Claim 8, including a construct according to Claim 5 or a vector according to Claim 6 or 7.

10. A method of modifying disease and/or pest resistance, plant defense 5 response and/or protein storage in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to any one of Claims 1-4, a construct according to Claim 5 and/or a vector according to Claim 6 or 7.

11. Use of a nucleic acid or nucleic acid fragment according to any one of Claims 1-4, and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof as a molecular genetic marker.

12. The use according to Claim 11 wherein the nucleic acid or nucleic acid fragment, nucleotide sequence information thereof and/or single nucleotide polymorphism thereof, is used as a molecular genetic marker for one or more of 15 quantative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting or market assisted selections.

13. The use according to Claim 11 or 12 wherein the molecular marker is used in Lolium species.

14. The use according to any one of Claims 11 to 13 wherein the molecular marker is used to select for disease and/or pest resistance, plant defense response and/or protein storage in a plant.

15. The use according to Claim 14 wherein the molecular marker is used to select for resistance to fungal or bacterial pathogens, virus resistance, insect resistance and/or nematode resistance. 25 16. A substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism from a nucleic acid or nucleic acid fragment according to any one of Claims 1 -4.

INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 AUG 2007 received 005014324 42

17 A substantially purified or isolated TH polypeptide from a Lolium species, or a functionally active fragment or variant thereof.

18. A polypeptide according to Claim 17, wherein said Lolium species is Lolium perenne or Lolium arundinaceum. 5

19. A substantially purified or isolated TH polypeptide including an amino acid sequence selected from the group consisting of sequences shown in Figures 2, 5, 8 and 36 hereto (Sequence ID Nos: 2, 21, 26 and 67, respectively): and functionally active fragments and variants thereof.

20. A polypeptide according to Claim 19 wherein said functionally active fragments and variants have at least approximately 95% identity with the relevant part of the recited sequences and have a size of at least 28 amino acids.

21. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding a polypeptide according to any one of Claims 17 to 20.

22. A nucleic acid or nucleic acid fragment according to Claim 1 substantially 15 as hereinbefore described with reference to any one of the figures or examples.

23. A polypeptide according to claim 17 substantially as hereinbefore described with reference to any one of the figures or examples. \ 7 AUG 2007 received