NZ529105A - An isolated nucleic acid or nucleic acid fragment encoding an abscisic acid-inducible and stress responsive (ASR) polypeptide for a ryegrass (lolium) species - Google Patents

Abstract

An isolated nucleic acid or nucleic acid fragment encoding an abscisic acid-inducible and stress responsive (ASR) polypeptide from a ryegrass (Lolium) species. Also disclosed is a method of modifying a plant response to an environmental stimulus by introducing into the plant an effective amount of nucleic acid. Also disclosed is the use of a nucleic acid as a molecular genetic marker.

Description

lu/uj/zuuo u j# : vj ^ 004785492 1 529105 MODIFICATION OF PLANT AND SEED DEVELOPMENT AND PLANT RESPONSES TO STRESSES AND STIMULI This application is the parent of a divisional application entitled "Modification of plant and seed development and plant responses to stresses and stimuli (2)".

The present invention relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for abscisic acid- and stress- inducible proteins, 5 cysteine proteases, late embryogenesis abundant proteins, dehydrins and protein kineses in plants and the use thereof for, inter alia, modification of plant tolerance to abiotic environmental stresses and osmotic stresses such as drought stress and salt 10 stress; modification of adaptation to temperature stresses such as plant cold acclimation, modification of seed development and/or germination in plants, 10 modification of plant responses to adverse environmental stimuli and/or modification of plant developmental processes.

The plant hormone abscisic acid (ABA) appears to influence several 15 physiological and developmental events. ABA plays a major role in the adaptation to abiotic environmental stresses, such as drought and high- salinity, as well as in 15 seed maturation and dormancy, and involves the expression of ABA-inducible and stress-responsive proteins (ASR and A22), which may function in dehydration tolerance in both vegetative tissues and seeds.

As a response to water deficit, there is an increase in the endogenous ABA levels, that rapidly limits water loss through transpiration by reducing stomatal 20 aperture. ABA is also involved in other aspects of stress adaptation such as cold acclimation and root morphogenesis in response to stress.

Many of the genes that are involved in ABA signal transduction pathways are 25 also induced by environmental stresses. They include stress- inducible cysteine proteases (CYS), late embryogenesis abundant proteins (LEA), 25 dehydrins (DHN) and ABA-induced protein kineses (PKABA).

INTELLECTUAL PROPERTY OFFiCE OF N.Z. 1 MAR 2008 ftEOEIVBO 1VJ/UJ/ £UUO VI 9 ' U £ 004785492 1a While nucleic acid sequences encoding some ASR, A22, CYS, LEA, DHN and PKABA have been isolated for certain species of plants, there remains a need for materials useful in modifying plant tolerance to abiotic environmental stresses and osmotic stresses such as drought stress and salt stress; modifying adaptation 30 to 5 temperature stresses such as plant cold acclimation, modifying seed 2 development and/or germination in plants, modifying plant responses to adverse environmental stimuli, and/or modifying plant developmental processes, in a wide range of plants, particularly in forage and turf grasses and legumes, including ryegrasses, fescues and clovers, and for methods 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 isolated nucleic acids or nucleic acid fragments encoding ASR, A22, CYS, LEA, DHN and PKABA from a ryegrass (Lolium) or fescue (Festuca) species and 10 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 proteins which are related to ASR, A22, CYS, LEA, DHN and PKABA and functionally active fragments and variants thereof. Such proteins are referred to 15 herein as ASR-like, A22-like, CYS-like, LEA-like, DHN-like and PKABA-like, respectively.

The individual or simultaneous enhancement or otherwise manipulation of ASR, A22, CYS, LEA, DHN and/or PKABA or like gene activities in plants may enhance or otherwise alter plant tolerance to abiotic environmental stresses, for 20 example tolerance to drought stress; may enhance or otherwise alter plant tolerance to osmotic stress, for example to salt stress; may enhance or otherwise alter adaptation to temperature stress, for example cold acclimation; may enhance or reduce or otherwise alter seed development, for example seed maturation; may enhance or reduce or otherwise alter seed germination, for example seed 25 dormancy; and enhance or otherwise alter plant responses to adverse environmental stimuli, for example dehydration; or may alter plant developmental processes, for example root morphogenesis.

The individual or simultaneous enhancement or otherwise manipulation of ASR, A22, CYS, LEA, DHN and/or PKABA or like gene activities in plants has 3 significant consequences for a range of applications in, for example, plant production and plant protection. For example, it has applications in increasing plant tolerance to abiotic environmental stresses such as water stress; in increasing plant tolerance to osmotic stresses such as salt stress; in increasing 5 plant tolerance to temperature stresses such as cold stress; in increasing the spectrum of abiotic stress tolerance to a wide range of environmental stresses; in reducing plant damage caused by environmental stresses such as dehydration and cold; in improving biomass productivity under conditions of abiotic environmental stress such as water deficient conditions; in improving water use 10 efficiency under conditions of abiotic environmental stress such as water deficient conditions; in helping to protect plant cells against dehydration; in altering metabolism by increasing protein turnover rates or proteolytically activating specific proteins; in degrading polypeptides denatured because of cellular stress or of storage proteins leading to mobilisation of amino acids which would be 15 available for the synthesis of new proteins in response to stress or for osmotic adjustment; in altering seed maturation and/or seed dormancy and in altering plant developmental processes.

Methods for the manipulation of ASR, A22, CYS, LEA, DHN and/or PKABA or like gene activities in plants, including grass species such as ryegrasses 20 (Lolium species) and fescues (Festuca species), and legumes such as clovers {Trifolium species) may facilitate the production of, for example, pasture and turf grasses and pasture legumes with enhanced tolerance to abiotic stresses such as drought, or enhanced tolerance to salt stress, or enhanced tolerance to temperature stresses such as cold, or modified seed maturation, or modified seed 25 germination, or modified plant developmental processes such as root morphogenesis.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably 30 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. 4 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 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 10 nucleic acids or nucleic acid fragments could be part of a vector and/or such nucleic acids or nucleic acid fragments could be part of a composition, and still be isolated in that 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 15 nucleotide sequence that is assembled from two or more constituent nucleotide sequences that 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 acid 20 fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an ASR or ASR-like protein includes a nucleotide sequence selected from the group consisting of 25 (a) sequences shown in Figures 1, 3 and 34 hereto (Sequence ID Nos: 1, 3 to 15, 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).

In a further preferred embodiment of this aspect of the invention, the 30 substantially purified or isolated nucleic acid or nucleic acid fragment encoding an A22 or A22-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 4, 6, 7, 9, 10, 12 and 27 hereto (Sequence ID Nos: 16,18 to 21, 22, 24 to 28, 29, 31 to 34, and 64, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the 5 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 CYS or CYS-like protein includes a nucleotide sequence selected from the group 10 consisting of (a) sequences shown in Figures 13,15, 41 and 47 hereto (Sequence ID Nos: 35, 37 to 48, 68 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 LEA or LEA-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 16, 18 and 52 hereto (Sequence ID Nos: 49, 51 to 53, and 72, 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 25 DHN or DHN-like protein includes a nucleotide sequence selected from the group consisting of (a) sequence shown in Figure 19 hereto (Sequence ID No: 54); (b) complement of the sequence 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). 6 In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a PKABA or PKABA-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 21, 23, 25, 58 and 64 hereto 5 (Sequence ID Nos: 56, 58, 60 to 63, 74 and 76, 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).

By "functionally active" in relation to nucleic acids it is meant that the 10 fragment or variant (such as an analogue, derivative or mutant) is capable of modifying plant tolerance to abiotic environmental stresses and osmotic stresses such as drought stress and salt stress; modifying adaptation to temperature stresses such as plant cold acclimation, modifying seed development and/or germination in plants, modifying plant responses to adverse environmental stimuli 15 and/or modifying plant developmental processes, in a plant. Such variants include naturally occurring allelic variants and non-naturally occurring 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 20 has at least approximately 80% identity to the relevant part of the above mentioned sequence, more preferably at least approximately 90% identity, most 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 25 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.

Nucleic acids or nucleic acid fragments encoding at least a portion of several ASR, A22, CYS, LEA, DHN and PKABA have been isolated and identified. 30 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 7 protocols, such as 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 ASR or ASR-like, A22 or A22-like, CYS or CYS-like, LEA or LEA-like, DHN or DHN-like, and PKABA or PKABA-like proteins, either as cDNAs or genomic DNAs, may be 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 10 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. 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 15 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 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 20 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 nucleic acid fragments encoding homologous genes from DNA or RNA. For example, polymerase chain reaction may be performed on a library of cloned 25 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' end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For 30 example, those skilled in the art can follow the RACE protocol [Frohman et al. (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 8 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' cDNA fragments may be isolated [Ohara et al. (1989J Proc. Natl. Acad Sci USA 86:5673; Loh et al. (1989) Science 243:217; the entire disclosures of which are 5 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 purified or isolated polypeptide from a ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of ASR and ASR-like, A22 and A22-10 like, CYS and CYS-like, LEA and LEA-like, DHN and DHN-like and PKABA and PKABA-like proteins, and functionally active fragments and variants thereof.

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

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated ASR or ASR-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 2 and 35 hereto (Sequence ID Nos: 2 and 67, respectively) and functionally active 20 fragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated A22 or A22-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 5, 8, 11 and 28 hereto (Sequence ID Nos: 17, 23, 30 and 65, respectively) and 25 functionally active fragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated CYS or CYS-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures WO 02/090547 PCT/AU02/00564 9 14, 42 and 48 hereto (Sequence ID Nos: 36, 69 and 71, respectively) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated LEA or LEA-like polypeptide includes an amino 5 acid sequence selected from the group consisting of sequences shown in Figures 17 and 53 hereto (Sequence ID Nos: 50 and 73, respectively) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated DHN or DHN-like polypeptide includes an amino 10 acid sequence shown in Figure 20 hereto (Sequence ID No: 55) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated PKABA or PKABA-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in 15 Figures 22, 24, 59 and 65 hereto (Sequence ID Nos: 57, 59, 75 and 77, respectively) and functionally active fragments and variants thereof.

By "functionally active" in relation to polypeptides it is meant that the fragment or variant has one or more of the biological properties of the proteins ASR, ASR-like, A22, A22-like, CYS, CYS-like, LEA, LEA-like, DHN, DHN-like, 20 PKABA and PKABA-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% identity to the relevant part of the above mentioned sequence, more 25 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, more preferably at least 15 amino acids, 30 most preferably at least 20 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 10 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 in genotype are important in commercial breeding programs, in determining 15 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 allelic between organisms because it then may serve to unambiguously identify an 20 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 sequence information including single nucleotide polymorphisms (SNPs), 25 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 the black background) in the figures that show multiple alignments of nucleotide 30 sequences of nucleic acid fragments contributing to consensus contig sequences.

WO 02/090547 PCT/AU02/00564 11 See for example, Figures 3, 6, 9,12,15,18 and 25 hereto (Sequence ID Nos: 3 to 15,18 to 21,24 to 28, 31 to 34, 37 to 48, 51 to 53, and 60 to 63, respectively).

Accordingly, in a further aspect of the present invention, there is provided a substantially purified or isolated nucleic acid or nucleic acid fragment including a 5 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 of isolating a nucleic acid or nucleic acid fragment of the present invention 10 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 15 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 20 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 sequence information thereof, as a molecular genetic marker.

More particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, 12 DNA fingerprinting and in marker assisted selection, particularly in ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers in forage and turf grass improvement in 5 relation to plant tolerance to abiotic, osmotic or temperature stresses, response to environmental stimuli, and/or seed development and/or germination and/or plant developmental processes such as root morphogenesis, e.g. tagging QTLs for tolerance to drought, for tolerance to salt, for tolerance to cold, and for seed dormancy. Even more particularly, sequence information revealing SNPs in allelic 10 variants of the nucleic acids or nucleic acid fragments of the present invention and/or nucleotide sequence information thereof 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 15 including a nucleic acid or nucleic acid fragment according to the present 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 20 cases can also be the gene of interest and appropriate regulatory sequences. It 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 present invention there is provided a vector including a 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 30 from several sources. In a preferred embodiment of this aspect of the invention, 13 the vector may 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 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 10 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 viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium 15 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 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 which may be employed in the vectors of the present invention are well known to 25 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 the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin 30 promoter, and the rice Actin promoter.

WO 02/090547 PCT/AU02/00564 14 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. Particularly suitable terminators are polyadenylation signals, such as the CaMV 5 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 fragments of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid or nucleic acid fragments, in 10 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 genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or 15 pat) gene and the gentamycin acetyl transferase (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 appropriate sequences for amplifying expression.

As an alternative to use of a selectable marker gene to provide a 20 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, histochemical GUS assays, northern and Western blot hybridisation analyses.

Those skilled in the art will appreciate that the various components of the 25 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 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 turfgrasses, corn, oat, sugarcane, wheat and barley), dicotyledons (such as 5 Arabidopsis, tobacco, white clover, red clover, subterranean clover, alfalfa, eucalyptus, potato, sugarbeet, 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) and fescues (Festuca species), even more preferably perennial ryegrass, 10 including forage- and turf-type cultivars. In an alternate preferred embodiment, the 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 15 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 20 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 30 asexually, using methods well known in the art, to produce successive generations of transformed plants. 16 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 5 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 ryegrass, including both forage- and turf-type cultivars. In an alternate preferred 10 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 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 20 modifying plant response to environmental stimulus, said method including introducing into said plant an effective amount of a nucleic acid or nuclei acid fragment, construct and/or a vector according to the present invention. Preferably, said environmental stimulus is selected from the group consisting of dehydration and cold.

In a further aspect of the present invention there is provided a method of modifying plant tolerance to abiotic, osmotic and/or temperature stresses said method including introducing into said plant an effective amount of a nucleic acid or nuclei acid fragment, construct and/or a vector according to the present WO 02/090547 PCT/AU02/00564 17 invention. Preferably, said stress is selected from the group consisting of water stress, salt stress and cold stress.

In a further aspect of the present invention there is provided a method of modifying seed development, maturation, dormancy and/or germination in a plant, 5 said method including introducing into said plant an effective amount of a nucleic acid or nuclei acid fragment, construct and/or a vector according to the present invention.

In a further aspect of the present invention there is provided a method of modifying a plant developmental process, said method including introducing into 10 said plant an effective amount of a nucleic acid or nuclei acid fragment, construct and/or a vector according to the present invention. Preferably said developmental process is root morphogenesis.

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 15 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 suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring 20 Harbor, the entire disclosure of which is incorporated herein by reference.

Using the methods and materials of the present invention, tolerance to abiotic, osmotic or temperature stresses, plant response to environmental stimuli, seed maturation and/or germination, and/or plant developmental processes may be increased, decreased or otherwise modified relative to an untransformed 25 control plant. For example, response to environmental stimuli; tolerance abiotic, osmotic and/or temperature stresses such as drought stress, salt stress and/or cold; seed maturation and/or germination; and/or plant developmental processes such as root morphogenesis, may be increased or otherwise altered, for example, by incorporating additional copies of a sense nucleic acid or nucleic acid fragment 30 of the present invention. They may be decreased or otherwise modified, for 18 example, by incorporating an antisense 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 5 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 LpASR (Sequence ID No: 1).

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

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

Figure 4 shows the consensus contig nucleotide sequence of LpA22a (Sequence 15 ID No: 16).

Figure 5 shows the deduced amino acid sequence of LpA22a (Sequence ID No: 17).

Figure 6 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpA22a (Sequence ID Nos: 18 to 20 21).

Figure 7 shows the consensus contig nucleotide sequence of LpA22ab (Sequence ID No: 22).

Figure 8 shows the deduced amino acid sequence of LpA22ab (Sequence ID No: 23).

WO 02/090547 PCT/AU02/00564 19 Figure 9 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpA22ab (Sequence ID Nos: 24 to 28).

Figure 10 shows the consensus contig nucleotide sequence of LpA22ac 5 (Sequence ID No: 29).

Figure 11 shows the deduced amino acid sequence of LpA22ac (Sequence ID No: 30).

Figure 12 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpA22ac (Sequence ID Nos: 31 to 10 34).

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

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

Figure 15 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCYSa (Sequence ID Nos: 37 to 48).

Figure 16 shows the consensus contig nucleotide sequence of LpLEAa (Sequence ID No: 49).

Figure 17 shows the deduced amino acid sequence of LpLEAa (Sequence ID No: 50).

Figure 18 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpLEAa (Sequence ID Nos: 51 to 53).

Figure 19 shows the nucleotide sequence of LpDHNa (Sequence ID No: 54).

WO 02/090547 PCT/AU02/00564 Figure 20 shows the deduced amino acid sequence of LpDHNa (Sequence ID No: 55).

Figure 21 shows the nucleotide sequence of LpPKABAa (Sequence ID No: 56).

Figure 22 shows the deduced amino acid sequence of LpPKABAa (Sequence ID 5 No: 57).

Figure 23 shows the consensus contig nucleotide sequence of LpPKABAb (Sequence ID No: 58).

Figure 24 shows the deduced amino acid sequence of LpPKABAb (Sequence ID No: 59).

Figure 25 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPKABAb (Sequence ID Nos: 60 to 63).

Figure 26 shows a plasmid map of the cDNA encoding perennial ryegrass A22c.

Figure 27 shows the full nucleotide sequence of perennial ryegrass A22c cDNA 15 (Sequence ID No: 64).

Figure 28 shows the deduced amino acid sequence of perennial ryegrass A22c cDNA (Sequence ID No: 65).

Figure 29 shows plasmid maps of sense and antisense constructs of LpA22c in pDH51 transformation vector.

Figure 30 shows plasmid maps of sense and antisense constructs of LpA22c in pKYLX71:35S2 binary transformation vector.

Figure 31 shows plasmid maps of sense and antisense constructs of LpA22c in pPZP221:35S2 binary transformation vector.

WO 02/090547 PCT/AU02/00564 21 Figure 32 shows screening by Southern hybridisation for RFLPs using LpA22c as a probe.

Figure 33 shows a plasmid map of the cDNA encoding perennial ryegrass ASRa.

Figure 34 shows the full nucleotide sequence of perennial ryegrass ASRa cDNA 5 (Sequence ID No: 66).

Figure 35 shows the deduced amino acid sequence of perennial ryegrass ASRa cDNA (Sequence ID No: 67).

Figure 36 shows plasmid maps of sense and antisense constructs of LpASRa in pDH51 transformation vector.

Figure 37 shows plasmid maps of sense and antisense constructs of LpASRa in pKYLX71:35S2 binary transformation vector.

Figure 38 shows plasmid maps of sense and antisense constructs of LpASRa in pPZP221:35S2 binary transformation vector.

Figure 39 shows screening by Southern hybridisation for RFLPs using LpASRa as 15 a probe.

Figure 40 shows a plasmid map of the cDNA encoding perennial ryegrass CYSa.

Figure 41 shows the full nucleotide sequence of perennial ryegrass CYSa cDNA (Sequence ID No: 68).

Figure 42 shows the deduced amino acid sequence of perennial ryegrass CYSa 20 cDNA (Sequence ID No: 69).

Figure 43 shows plasmid maps of sense and antisense constructs of LpCYSa in pDH51 transformation vector. 22 Figure 44 shows plasmid maps of sense and antisense constructs of LpCYSa in pPZP221:35S2 binary transformation vector.

Figure 45 shows screening by Southern hybridisation for RFLPs using LpCYSa as a probe.

Figure 46 shows a plasmid map of the cDNA encoding perennial ryegrass CYSme.

Figure 47 shows the full nucleotide sequence of perennial ryegrass CYSme cDNA (Sequence ID No: 70).

Figure 48 shows the deduced amino acid sequence of perennial ryegrass CYSme 10 cDNA (Sequence ID No: 71).

Figure 49 shows plasmid maps of sense and antisense constructs of LpCYSme in pDH51 transformation vector.

Figure 50 shows plasmid maps of sense and antisense constructs of LpCYSme in pPZP221:35S2 binary transformation vector.

Figure 51 shows a plasmid map of the cDNA encoding perennial ryegrass LEAa.

Figure 52 shows the full nucleotide sequence of perennial ryegrass LEAa cDNA (Sequence ID No: 72).

Figure 53 shows the deduced amino acid sequence of perennial ryegrass LEAa cDNA (Sequence ID No: 73).

Figure 54 shows plasmid maps of sense and antisense constructs of LpLEAa in pDH51 transformation vector.

Figure 55 shows plasmid maps of sense and antisense constructs of LpLEAa in pPZP221:35S2 binary transformation vector.

WO 02/090547 PCT/AU02/00564 23 Figure 56 shows screening by Southern hybridisation for RFLPs using LpLEAa as a probe.

Figure 57 shows a plasmid map of the cDNA encoding perennial ryegrass PKABAa.

Figure 58 shows the full nucleotide sequence of perennial ryegrass PKABAa cDNA (Sequence ID No: 74).

Figure 59 shows the deduced amino acid sequence of perennial ryegrass PKABAa cDNA (Sequence ID No: 75).

Figure 60 shows plasmid maps of sense and antisense constructs of LpPKABAa 10 in pDH51 transformation vector.

Figure 61 shows plasmid maps of sense and antisense constructs of LpPKABAa in pPZP221:35S2 binary transformation vector.

Figure 62 shows screening by Southern hybridisation for RFLPs using LpPKABAa as a probe.

Figure 63 shows a plasmid map of the cDNA encoding perennial ryegrass PKABAb.

Figure 64 shows the full nucleotide sequence of perennial ryegrass PKABAb cDNA (Sequence ID No: 76).

Figure 65 shows the deduced amino acid sequence of perennial ryegrass 20 PKABAb cDNA (Sequence ID No: 77).

Figure 66 shows plasmid maps of sense and antisense constructs of LpPKABAb in pDH51 transformation vector.

Figure 67 shows plasmid maps of sense and antisense constructs of LpPKABAb in pPZP221:35S2 binary transformation vector.

WO 02/090547 PCT/AU02/00564 24 Figure 68 shows screening by Southern hybridisation for RFLPs using LpPKABAb as a probe.

Figure 69 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 abiotic stress protection.

Figure 70 shows the genetic map detailing the relation of perennial ryegrass genes involved in abiotic stress protection with the linkage groups in perennial 10 ryegrass.

Figure 71 shows a subgrid of a microarray for the expression profiling of perennial ryegrass genes involved in abiotic stress protection.

EXAMPLE 1 Preparation of cDNA libraries, isolation and sequencing of cDNAs coding for ASR, ASR-like, A22, A22-like, CYS, CYS-like, LEA, LEA-like, PKABA and PKABA-like proteins from perennial ryegrass (Lolium perenne) cDNA libraries representing mRNAs from various organs and tissues of perennial ryegrass (Lolium perenne) were prepared. The characteristics of the libraries are described below (Table 1).

WO 02/090547 PCT/AU02/00564 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) 05 rg Senescing leaves from mature plants 06 rg Whole etiolated seedlings (1-5 day old and 17 days old) 07rg Roots from mature plants grown in hydroponic culture 08rg Senescent leaf tissue 09 rg 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) 26 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 0 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 0 plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut pBluescript 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 5 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 0 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 ABI 3700 sequence analyser. wo 02/090547 27 pct/au02/00564 EXAMPLE 2 DNA sequence analyses The cDNA clones encoding ASR, ASR-like, A22, A22-like, CYS, CYS-like, 5 LEA, LEA-like, DHN, DHN-like, PKABA and PKABA-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 10 National Center for Biotechnology Information (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 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 20 consensus contig sequence was then used as a query for a search against the SWISS-PROT protein sequence database using the BLASTx algorithm to confirm the initial identification.

EXAMPLE 3 Identification and full-length sequencing of cDNAs encoding perennial 25 ryegrass ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb 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 ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb proteins was identified and fully sequenced. 28 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 using Sequencher (Gene Codes Corp., Ann Arbor, Ml 48108, USA). The original 5 plasmid was then used to transform chemically competent XL-1 cells (prepared in-house, CaCl2 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 with the original EST sequence using Sequencher to confirm identity and one of 10 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 cases the sequencing could be done from both 5' and 3' end. The sequences of 15 the 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 sequences of the SMART primers used to generate the initial cDNA library as well 20 as pGEM-T Easy vector sequence up to the EcoRI cut site both at the 5' and 3' end.

Plasmid maps and the full cDNA sequences of perennial ryegrass cDNAs encoding ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb proteins were obtained (Figures 26, 27, 33, 34, 40, 41,46,47, 51, 52, 57, 58, 63, and 64). wo 02/090547 pct/au02/00564 29 TABLE 2 List of primers used for sequencing of the full-length cDNAs gene name clone ID sequencing primer primer sequence (5'>3') LpA22c 14rg1ZsA09 14rg1ZsA09.f1 tcaacatttggtatgtccc LpCYSa 08rg2AsA02 08rg2AsA02.f1 aagacgtcgcgccgctcg 08rg2AsA02.f2 acggtgttcttctagttgg LpLEAa 11rg1GsB02 11rg1GsB02.f1 atgcgacttgatccttgc LpPKABAa 20rg2CsF06 20rg2CsF06.f1 tgtcttgcattctcaacc LpPKABAb 16rg1 DsG05 16rg1DsG05.f1 tgttttgcactctcagcc 16rg1DsG05.f2 ttcgcagggcagctttcc EXAMPLE 4 Development of transformation vectors containing chimeric genes with ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb cDNA sequences from perennial ryegrass To alter the expression of the proteins involved in abiotic stress protection ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb, through antisense 10 and/or sense suppression technology and for over-expression of these key enzymes 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 15 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 into the corresponding site in pDH51, a pUC18-based transformation vector containing a CaMV 35S expression cassette. The orientation of the constructs 20 (sense or antisense) was checked by DNA sequencing through the multi-cloning site of the vector. Transformation vectors containing chimeric genes using full- WO 02/090547 PCT/AU02/00564 length open reading frame cDNAs encoding perennial ryegrass ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb proteins in sense and antisense orientations under the control of the CaMV 35S promoter were generated (Figures 29,36,43,49, 54, 60 and 66).

TABLE 3 List of primers used to PCR-amplify the open reading frames gene name clone ID primer primer sequence (5'->3') LpA22c 14rg1ZsA09 14rg1ZsA09.f gaattctagattcgaggaaagaggagcaggcatgg 14rg1ZsA09.rl gcattctagatgcgttggtcagtcttggatgg LpASRa 18rg1AsE01 18rg1AsE01.f gcattctagatagctagctcagcagatcagtcgg 18rg1AsE01.r gcattctagaacacgtagaaaaagcacacgggcg LpCYSa 08rg2AsA02 08rg2AsA02.f gaattctagaaagaaatccgccgatggatcgtcg 08rg2AsA02.r gaattctagattcgcatcttcgctgatcatacc LpCYSme 07rglQsD02 07rg1QsD02.f gaattctagatcgtcttaccgtttactcagaccg 07rg1QsD02.r gaattctagaatctgcataagcttgcacagttgg LpLEAa 11 rg1 GsB02 11rg1GsB02.f gaattctagaatcgagtggtcagatcaaaagagc 11rg1GsB02.r gaattctagaactgattcagctaacaagtgacc LpPKABAa 20rg2CsF06 20rg2CsF06.f gacgtcgacaaagggatggatcggtacgagg 20rg2CsF06.r gacgtcgacgctacttttgaaccactcgtgacc LpPKABAb 16rg1DsG05 16rg1DsG05.f gacgtcgacatccagtgccgattacgagctgcg 16rg1DsG05.r gacgtcgaccctcgcctgaaccacacctgc EXAMPLE 5 Development of binary transformation vectors containing chimeric genes 10 with ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb cDNA sequences from perennial ryegrass To alter the expression of the proteins involved in abiotic stress protection ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb, through antisense and/or sense suppression technology and for over-expression of these key WO 02/090547 PCT/AU02/00564 31 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 5 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 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 10 gene nptll under the control of the nos promoter and nos terminator and an 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 15 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 EcoRI, 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-20 cloning site was removed by digestion with EcoRI, and the expression cassette cloned into the EcoRI 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 with a 25 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 ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb in sense and antisense orientations under 30 the control of the CaMV 35S2 promoter were generated (Figures 30, 31, 37, 38, 44,50, 55, 61 and 67). 32 EXAMPLE 6 Production and analysis of transgenic Arabidopsis plants carrying chimeric perennial ryegrass genes ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and 5 PKABAb involved in abiotic stress protection A set of transgenic Arabidopsis plants carrying chimeric perennial ryegrass genes involved in abiotic stress protection were produced.

PPZP221-based transformation vectors with LpASRa, LpA22c, LpCYSa, LpCYSme, LpLEAa , LpPKABAa and LpPKABAb cDNAs comprising the full open 10 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 these transformation vectors.

The production of transgenic Arabidopsis plants carrying the perennial ryegrass ASRa, A22c, CYSa, CYSme, LEAa, PKABAa and PKABAb cDNAs 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 thaliana ecotype Columbia, Lehle Seeds #WT-02) sown to obtain approximately 15 plants per punnet. The seeds were then 25 vernalised by placing the punnets at 4 SC. After 48 hours the punnets were transferred to a growth room at 22 SC under fluorescent light (constant illumination, 55 /vmolm"2s"1) and fed with Miracle-Gro (Scotts Australia Pty. Ltd.) 33 once a week. Primary bolts were removed as soon as they appeared. After 4-6 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 /vg/ml rifampicin and 50 /vg/ml kanamycin and grown at 27 eC for 48 hours. A single colony was used to inoculate 5 ml of LB medium containing 50 /vg/ml rifampicin and 50 /vg/ml kanamycin and grown over night at 27 9C and 250 rpm on an orbital shaker. The overnight culture was used as an inoculum for 500 10 ml of LB medium containing 50 //g/mf kanamycin only. Incubation was over night at 27 eC and 250 rpm on an orbital shaker in a 2 I Erlenmeyer flask.

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)] 15 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 20 the Agrobacterium suspension and a gentle vacuum (250 mm Hg) was applied for 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 25 stored at room temperature in a dry container or used for selection of Selection of transformants 34 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 /vl 5 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 fj\ 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 /vl 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 /vg/ml timetin and 75 /vg/ml gentamycin. After vernalisation for 48 hours at 4 QC the plants were grown under continuous fluorescent light (55 15 //mol m-2s-1) at 22 BC to the 6 - 8 leaf stage and transferred to soil.

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 20 DNA was isolated from the supernatant using Wizard Magnetic 96 DNA Plant System kits (Promega) on a Biomek FX (Beckman Coulter). 5 /vl of the sample (50 /vl) 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 WO 02/090547 PCT/AU02/00564 within the 35S2 promoter region guaranteed that homologous genes in Arabidopsis were not detected. fj\ of each genomic DNA sample was run in a 50 //I PCR reaction including SYBR Green on an ABI (Applied Biosystems) together with samples 5 containing DNA isolated from wild type Arabidopsis plants (negative control), 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 10 representative real-time PCR analyses are shown in Figure 69.

TABLE 4 List of primers used for Real-time PCR analysis of Arabidopsis plants transformed with chimeric perennial ryegrass genes involved in abiotic stress protection construct primer 1 (forward) primer 2 (reverse) LpA22csense TTGGAGAGGACACGCTGAAATCAC GCCCTGCAAGAACATCGAAGTTC LpA22canti TCATTTCATTTGGAGAGGACACGC TCAGATTATCACAGCACATACCCCG LpASRasense TTGGAGAGGACACGCTGAAATCAC CCCTCCTTCTTGTGGTGAAACAGG LpASRaanti rCATTTCATTTGGAGAGGACACGC CACGAGAAGAAGGAGGACCACAAG LpCYSasense TCATTTCATTTGGAGAGGACACGC CGTTGCCGACGACCTGCC LpCYSaanti TTGGAGAGGACACGCTGAAATCAC TGCCATTAACGCCTCAGAGGAGTAG LpCYSmesense TCATTTCATTTGGAGAGGACACGC TTGATGCCGCCGACCAGC LpCYSmeanti TTGGAGAGGACACGCTGAAATCAC TGTGGGAGAAGCAATGGGAGAAC LpLEAasense TCATTTCATTTGGAGAGGACACGC CCTGGACCTTCGCTTTGATGG LpLEAaanti TGACGTAAGGGATGACGCACAATC TGCCTTTAGAGCTAGTCCTGTTGGG LpPKABAasense AATCCCACTATCCTTCGCAAGACC GCCTCTCTCGATGAACTTGACGG LpPKABAaanti ATTTCATTTGGAGAGGACACGCTG AGGTTGCCAGCCTGGGGAG LpPKABAbsense AATCCCACTATCCTTCGCAAGACC AGCTCCTTGGTCCTGATGTCCC LpPKABAbanti TTGGAGAGGACACGCTGAAATCAC AGTTCGCAGGGCAGCTTTCC 36 EXAMPLE 7 Genetic mapping of perennial ryegrass genes involved in abiotic stress protection The cDNAs representing genes involved in abiotic stress protection were 5 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 cultivar Aurora, which is derived from a Swiss 10 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 DNA (10 jxg) of both parents and 5 Fi progeny restricted with the enzymes Dral, EcoRI, EcoRV or Hindlll. Hybridisations were carried out using the method of 15 Sharp et al. (1988). Polymorphic probes were screened on a progeny set of 114 individuals restricted with the appropriate enzyme (Figures 32, 39, 45, 56, 62 and 68).

RFLP bands segregating within the population were scored and the data was entered into an Excel spreadsheet. Alleles showing the expected 1:1 ratio 20 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 ordered within each linkage group using a LOD threshold of 2.0.

Loci representing genes involved in abiotic stress protection mapped to the 25 linkage groups as indicated in Table 5 and in Figure 70. These gene locations can now be used as candidate genes for quantitative trait loci associated with plant tolerance to abiotic environmental stresses and osmotic stresses such as drought stress and salt stress; adaptation to temperature stresses such as plant cold acclimation, seed development and/or germination in plants, plant responses to 30 adverse environmental stimuli and/or plant developmental processes. 37 TABLE 5 Map locations of ryegrass genes encoding proteins involved in plant and seed development and plant responses to stresses and stimuli Probe Polymorphic Mapped Locus Linkage with group NA6 AU6 LpASRa Y Oral LpASRa 4 LpASRal Y EcoRI LpASRal 4 LpASRa2 Y Dra 1 LpASRa2 4 LpA22a Y EcoRV LpA22a.1 7 LpA22a.2 7 7 LpA22b Y Oral LpA22b '8' 2 LpA22c Y EcoRV LPA22C 4 LpCYSa Y Dra 1 LpCYSa 7 LpCYSme Y Hind III LpCYSme 3 LpLEAa Y EcoRI LpLEAa 7 LpDHNa Y Hind\\\ LpDHNa. 1 6 LpDHNa.2 6 LpPKABAb Y Dra 1 LpPKABAb 2 2 EXAMPLE 8 Expression profiling of cDNAs encoding proteins involved in abiotic stress protection using microarray technology cDNAs encoding proteins involved in abiotic stress protection were PCR 10 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 15 reversed transcribing RNA and incorporating Cyanine 3 or 5 labelled dCTP. The probes were hybridised onto microarrays. In each case the experiment was repeated on two microarrays. After hybridisation for 16 hours (overnight), the WO 02/090547 PCT/AU02/00564 38 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 2000 across all experiments and judged as not present (-), low expression (+), medium expression (++), high 5 expression (+++) and highly expression (++++) (Table 7).

TABLE 6 List of hybridization probes used in expression profiling of perennial ryegrass genes encoding proteins involved in plant and seed development and plant responses to stresses and stimuli Hybridisation probe for microarrays Organ specificity (3-months old plants grown hydroponically) Leaf blade 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) TABLE 7 Results of expression profiling of ryegrass genes encoding proteins involved in plant and seed development and plant responses to stresses and stimuli Clone name in patent Clone id Gene id length sheath leaf root Seed 5LS 7LS 10LS 5LR 7LR 10LR 5DS 7DS 10DS 5DR 7DR 10DR ASRal 07rgtRsD04 DsrASR2_LYCES12600 full ++ ++++ ++ + ++++ ++ ++++ ++++ ++++ ++++ ++++ ++++ ++ ++++ ++++ ++++ A22a 19rg1NsA01 ZunDP1_HUMAN-22244 full + + + ++ + + + ++ + ++ ++ + - ++ + + A22b 05rg1BsF01 ZunDP1_HUMAN-10735 full? + ++ + + + + + +++ ++ ++ ++ + + ++ ++ +++ A22c 14rg1ZsA09 BotDP 1 _MO US E-19950 full ++ + - + - ++ + - - - + + + - - - LEAa 11rg1YsF06 DsrLE25_LYC ES17141 full + + + ++ - + + + + + + - - - + + 40 REFERENCES An, G., Watson, B.D., Stachel, S., Gordon, M.P., Nester, E.W. (1985) New cloning 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 et al. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. 10 Acad Sci. USA 85:8998 Gish and States (1993) Identification of protein coding regions by database similarity search. Nature Genetics 3:266-272 Lander, E.S., Green P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E., Newburg, L. (1987). MAPMAKER: an interactive computer package for 15 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 chain. Science 243:217-220 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. Cold Spring Harbour Laboratory Press Schardl, C.L., Byrd, A.D., Benzion, G., Altschuler, M.A., Hildebrand, D.F., Hunt, 25 A.G. (1987) Design and construction of a versatile system for the expression WO 02/090547 PCT/AU02/00564 41 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 5 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 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 acknowledgment that they form part of the common general knowledge in the relevant art.

VJ / U J / £UUO 004785492 «J a . \j i. 42

Claims (26)

1. An isolated nucleic acid or nucleic acid fragment encoding an abscisic acid-inducible and stress responsive (ASR) polypeptide from a ryegrass (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 ryegrass species is perennial ryegrass (Lolium perenne).

3. An isolated nucleic acid or nucleic acid fragment encoding an ASR protein or complementary or antisense to a sequence encoding an ASR protein and including a nucleotide sequence selected from the group consisting of (a) sequences shown in 10 Figures 1, 3 and 34 hereto (Sequence ID Nos: 1, 3 to 15, and 66, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in 15 (a) and (b); and (d) functionally active fragments and variants having at least approximately 80% identity with the sequences recited in (a), (b) and (c).

4. A construct including a nucleic acid or nucleic acid fragment according to any one 15 ofClaims1-3.

5. A vector including a nucleic acid or nucleic acid fragment according to any one of Claims 1-3.

6. A vector according to Claim 5, further including a promoter and a terminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked. 20

7. A plant cell, plant, plant seed or other plant part, including a construct according to Claim 4 or a vector according to Claim 5 or 6.

8. A plant, plant seed or other plant part derived from a plant cell or plant according to Claim 7.

9. A method of modifying plant response to an environmental stimulus, said method 25 including introducing into said plant an effective amount of a nucleic acid or nucleic acid iNTtlLFCTlJAL PROPFRTY OFFSCE OF N.Z. 1 0 MAR 2006 RECEIVED 1U/U3/ZUU& 004785492 u»:uz 43 fragment according to any one of Claims 1-3, a construct according to Claim 4, and/or a vector according to Claim 5 or 6.

10. A method according to Claim 9, wherein said environmental stimulus is selected from the group consisting of dehydration and cold. 5

11. A method of modifying plant tolerance to abiotic, osmotic and/or temperature stresses, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to any one of Claims 1-3, a construct according to Claim 4, and/or a vector according to Claim 5 or 6.

12. A method according to Claim 11, wherein said stress is selected from the group 10 consisting of water stress, salt stress and cold stress.

13. A method of modifying seed development, maturation, dormancy and/or germination 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 1-3, a construct according to Claim 4, and/or a vector according to Claim 5 or 6. 15

14. A method of modifying a plant developmental process, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to any one of Claims 1-3, a construct according to Claim 4, and/or a vector according to Claim 5 or 6.

15. A method according to Claim 14, wherein said developmental process is root 20 morphogenesis.

16. Use of a nucleic acid or nucleic acid fragment according to any one of Claims 1-3, and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof as a molecular genetic marker.

17. The use according to Claim 16 wherein the molecular genetic marker for one or 25 more quantative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting or marker assisted selections. f <N1.:UEC7'JAL PROFl-ory OFFICE OF N.Z 1 0 ttAR 2006 RSCEIVEn 004844239 44

18. The use according to claim 16 or 17 wherein the molecular marker is used in ryegrass species.

19. The use according to any one of claims 16 to 18 wherein the molecular marker is used to select for plant tolerance to abiotic, osmotic or temperature stresses, response 5 to environmental stimuli, seed development, maturation, dormancy or germination, and/or plant developmental processes.

20. The use according to claim 19 wherein the molecular marker is used to select for tolerance to drought, salt or cold, and/or root morphogenesis.

21. A substantially purified or isolated nucleic acid or nucleic acid fragment including 10 a single nucleotide polymorphism from a nucleic acid or nucleic acid fragment according to any one of claims 1-3.

22. An isolated ASR polypeptide from a ryegrass (Lolium) species, and functionally active fragments and variants thereof.

23. A polypeptide according to Claim 22, wherein said ryegrass is perennial ryegrass 15 (Lolium perenne).

24. An isolated ASR polypeptide including an amino acid sequence selected from the I group consisting of sequences shown in Figures 2 and 35 hereto (Sequence ID Nos: 2 and 67, respectively); and functionally active fragments and variants thereof having at least approximately 80% identity with said sequences. 20

25. A nucleic acid or nucleic acid fragment according to claim 1 substantially as hereinbefore described with reference to any one of the examples.

26. A polypeptide according to claim 22 substantially as hereinbefore described with reference to any one of the examples. 1 s JUL 2006 / -S^CE/VED/