AU2010202793B2

AU2010202793B2 - Plant disease/pest resistance and defense response (2)

Info

Publication number: AU2010202793B2
Application number: AU2010202793A
Authority: AU
Inventors: Michael Emmerling; Eng Kok Ong; Timothy Ivor Sawbridge; German Spangenberg
Original assignee: Agriculture Victoria Services Pty Ltd
Current assignee: Agriculture Victoria Services Pty Ltd
Priority date: 2003-02-11
Filing date: 2010-07-01
Publication date: 2012-03-15
Anticipated expiration: 2024-02-10
Also published as: AU2003902424A0; AU2010202793A1; AU2004200482B2; NZ531107A; NZ541768A; NZ552985A; AU2004200482A1

Abstract

The present invention relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for proteinase inhibitors in plants and the use thereof for, inter alia, modification of plant defense response, disease and/or pest 5 resistance, manipulation of protein breakdown in plants. The present invention also relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for glucanases, chitinases and related proteins in plants and the use thereof for, inter alia, modification of plant defense response, disease and/or pest resistance, manipulation of glucan and/or chitin 10 breakdown, production of signals and elicitors for the manipulation of plant development, and/or host defense reaction in plants.

Description

P/Do/oil Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Plant disease/pest resistance and defense response (2) The following statement is a full description of this invention, including the best method of performing it known to us: 1A PLANT DISEASE/PEST RESISTANCE AND DEFENSE RESPONSE (2) This application is a divisional of Australian patent application no. 2004200482, the entire disclosure of which is incorporated herein by reference. The present invention relates to nucleic acids and nucleic acid fragments 5 encoding amino acid sequences for proteinase inhibitors in plants and the use thereof for, inter alia, modification of plant defense response, disease and/or pest resistance, manipulation of protein breakdown in plants. The present invention also relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for glucanases, chitinases and related 10 proteins in plants and the use thereof for, inter alia, modification of plant defense response, disease and/or pest resistance, manipulation of glucan and/or chitin breakdown, production of signals and elicitors for the manipulation of plant development, and/or host defense reaction in plants. Plant proteinase inhibitors (PI) are polypeptides or proteins that occur 15 naturally in a wide range of plants and are a part of the plant's natural defense system against herbivory. They inhibit digestive proteinases in for example, insects and nematodes, leading to growth and development retardation and death from starvation. Proteinases in insects include serine, cysteine, metallo and aspartic proteases that catalyse the breakdown of dietary protein and the release 20 of amino acids, and so provide the nutrients essential for normal insect growth and development. Different proteinases predominate in different insects, for example, serine proteinases are dominant in lepidopteran larvae while coleopteran species have a wider range of dominant gut proteases including particularly cysteine-proteinases. 25 Serine- and cysteine-proteinase inhibitors inhibit growth and development of a range of insect pests, mainly lepidopteran and coleopteran species, respectively. Proteinase inhibitors also affect water balance, moulting and enzyme regulation of insects. Plants have evolved a range of biochemical responses to defend against 30 challenging pathogens. The plant defense response is accompanied by the accumulation of a characteristic set of proteins, called 'pathogenesis-related' (PR) 2 proteins. PR proteins having known enzymatic function are glucanases (a-1,3 glucanases and p-1,3-glucanases) (GLUC) and chitinases (CHIT). These proteins represent potential antifungal hydrolases that can act synergistically to inhibit fungal growth in vitro and in planta. p-1,3-glucanases participate in the breakdown 5 of p--1,3-glucans present in the cell walls of phytopathogenic fungi or in different plant structures. Chitinases are involved in the breakdown of chitin. Chitin, a homopolymer of p-1,4-linked N-acetyl-D-glucosamine residues, is found in many invertebrates (in exoskeleton of arthropods and nematodes) and fungi. Glucan and chitin fragments released by the action of glucanases and 10 chitinases, respectively can act as elicitors of host defense reaction or signal molecules in normal plant development. Hydrolases, chitinases and glucanases are not only induced in a non specific form in plants undergoing stress, such as infection with fungi, bacteria, viruses, viroids, etc., but are also expressed in healthy roots, lower leaves, floral 15 organs and in vitro plant cell cultures. The enzymes can also be induced by wounding, exposure to ethylene, fungal cell wall preparations, environmental changes (e.g. ozone, UV irradiation) and abiotic elicitors such as salicylic acid and mercuric chloride. While nucleic acid sequences encoding some proteinase inhibitors 20 chitinases and glucanases 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, protein breakdown, glucan and/or chitin breakdown, production of signals and elicitors for the manipulation of plant development, and/or host defense reaction, in a wide range of plants, particularly in forage and 25 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.

3 In one aspect, the present invention provides substantially purified or isolated nucleic acids and nucleic acid fragments encoding proteinase inhibitors (PI), glucanases (GLUC) and chitinases (CHIT) from a ryegrass (Lolium) or fescue (Festuca) species, and functionally active fragments and variants thereof. 5 The present invention also provides substantially purified or isolated nucleic acids and nucleic acid fragments encoding amino acid sequences for a class of proteins which are related to PI, GLUC and CHIT. Such proteins are referred to herein as PI-like, GLUC-like, and CHIT-like, respectively. The individual or simultaneous enhancement or otherwise manipulation of 10 PI gene activities in plants may enhance or otherwise alter plant defense response, for example plant defense against herbivore predators; may enhance or otherwise alter plant disease and/or pest resistance, for example the pest resistance status of plants to attack by pests such as insects or nematodes; may limit wounds and damage caused by plant diseases and/or pests; may limit the 15 spread of diseases and/or pests, including pathogens and other opportunistic microbes; may activate a range of cellular responses in the plant to minimize disease, pest/and or pathogen spread; or may alter protein breakdown during different plant developmental processes such as seed development and seed germination. 20 The modification of plant defense response based on the individual or simultaneous enhancement or otherwise manipulation of PI gene activities in plants has significant consequences for a range of applications in plant production and plant protection. For example, it has applications in increasing plant resistance to insect pests; in increasing plant resistance to nematode infection; in 25 reducing plant damage caused by herbivore predators; in reducing plant wounding caused by herbivore predators; in reducing entry points for and spread of pathogens; and in reducing the reliance on chemical applications eg. insecticides, nematicides, fungicides, etc. for pest and disease control. Methods for the modification of plant defense response, disease and/or pest resistance, 30 manipulation of protein breakdown in plants, including grass species such as ryegrasses (Lolium species) and fescues (Festuca species), and legumes such as 4 clovers (Trifolium species) 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, or modified plant defense response, or modified protein breakdown. 5 The individual or simultaneous enhancement or otherwise manipulation of GLUC and/or CHIT gene activities in plants may enhance or otherwise alter the disease resistance status of plants when challenged with pathogens such as fungi, bacteria, viruses or viroids; or may enhance or otherwise alter the pest resistance status of plants to attack by pests such as insects or nematodes; may limit the 10 spread of pathogens or pests or other opportunistic microbes; may activate a range of cellular responses in the plant to minimize pathogen, pest or disease spread; or may alter plant developmental processes such as reproductive development and embryogenesis. The modification of plant defense response based on the individual or 15 simultaneous enhancement or otherwise manipulation of GLUC and/or CHIT gene activities in plants has significant consequences for a range of applications in 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 insect pests; in increasing plant resistance to 20 nematode infection; in increasing spectrum of disease resistance to a wide range of pathogens; and in reducing the reliance on chemical applications eg. fungicides, insecticides, etc. for disease and pest control. Methods for the modification of plant defense response, disease and/or pest resistance, manipulation of glucan and/or chitin breakdown, and production of signals and elicitors for the 25 manipulation of plant development and/or host defense reaction in plants, including grass species such as ryegrasses (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 resistance to diseases and/or pests, or modified plant defense 30 response.

5 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 perennial ryegrass (L. perenne). Perennial ryegrass (Lolium perenne L.) is a key 5 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 10 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 present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the 15 natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids 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 20 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 25 acids or nucleic acid fragments, the sequences (and thus their corresponding nucleic acids or 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 a PI or PI-like 30 protein includes a nucleotide sequence selected from the group consisting of (a) 6 sequences shown in Figures 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 20, 21, 23, 24, 26 and 27 hereto (Sequence ID Nos. 1, 3-7, 8, 10-12, 13, 15-16, 17, 19-21, 22, 24-26, 27, 29, 31-37, 38, 40-58, 59, 61-62 and 63, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited 5 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 GLUC or GLUC-like protein includes a nucleotide sequence selected from the 10 group consisting of (a) sequences shown in Figures 29, 31, 33, 34, 36, 38, 40, 41, 43, 44, 46, 47, 49, 51, 53, 55, 56, 58, 60, 61 and 63 hereto (Sequence ID Nos. 65, 67, 69-77, 78, 80, 82, 84-85, 86, 88-90, 91, 93-95, 96, 98, 100, 102, 104-106, 107, 109, 111-115, 116 and 118, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and 15 (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 CHIT or CHIT-like protein includes a nucleotide sequence selected from the group 20 consisting of (a) sequences shown in Figures 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85 and 86 hereto (Sequence ID Nos. 120, 122-137, 138, 140-144, 145, 147-151, 152, 154-156, 157, 159-163, 164, 166-167, 168, 170-173 and 174, respectively; (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active 25 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 fragment or variant (such as an analogue, derivative or mutant) is capable of modifying one or more of the biological properties of the proteins PI and PI-like, GLUC and GLUC-like, and CHIT and CHIT-like, respectively, in a plant, such as 30 plant defense response, pest and/or disease resistance, protein breakdown, glucan or chitin breakdown, production of signals or elicitors for the manipulation 7 of plant development, and/or host defense reaction. 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 5 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 preferably at least approximately 95% identity. Such functionally active variants and fragments include, for example, those having nucleic acid changes which 10 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, more preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 45 nucleotides, most preferably at least 60 nucleotides. 15 The nucleic acids or nucleic acid fragments encoding at least a portion of several proteinase inhibitors, glucanases and chitinases have been isolated and identified. The nucleic acids and 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 20 dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, 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). 25 For example, genes encoding other proteinase inhibitors, glucanases or chitinases, 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 methodology well known to those skilled in the art. Specific 30 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 8 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 5 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 10 nucleic acids or 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 acids or nucleic acid fragments of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic 15 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 example, those skilled in the art can follow the RACE protocol (Frohman et al. (1988) Proc. Nat/. Acad Sci. USA 85:8998, the entire disclosure of which is incorporated herein by reference) to generate cDNAs by 20 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' 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 25 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 PI and PI-like, GLUC and GLUC like, CHIT and CHIT-like proteins; and functionally active fragments and variants 30 thereof.

9 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 perennial ryegrass (L. perenne). 5 In a preferred embodiment of this aspect of the invention, the substantially purified or isolated PI or Pt-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 2, 5, 8, 11, 14, 17, 19, 22, 25 and 28 hereto (Sequence ID Nos. 2, 9, 14, 18, 23, 28, 30, 39, 60 and 64, respectively); and functionally active fragments and variants thereof. 10 In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated GLUC or GLUC-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 30, 32, 35, 37, 39, 42, 45, 48, 50, 52, 54, 57, 59, 62 and 64 hereto (Sequence ID Nos. 66, 68, 79, 81, 83, 87, 92, 97, 99, 101, 103, 108, 110, 117 and 15 119, respectively); and functionally active fragments and variants thereof. In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated CHIT or CHIT-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 66, 69, 72, 75, 78, 81, 84 and 87 hereto (Sequence ID Nos. 121, 139, 146, 153, 20 158, 165, 169 and 175, 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 PI and PI-like GLUC and GLUC-like, CHIT and CHIT-like, respectively. Additions, 25 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 preferably at least approximately 80% identity, most 30 preferably at least approximately 90% identity. Such functionally active variants 10 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, most preferably at least 20 amino acids. 5 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 10 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 peptides and/or proteins comprising the amino acid sequences. These antibodies 15 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 parentage, in diagnostics and fingerprinting, and the like. Genotypes can be 20 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 individual. Furthermore, a genetic marker becomes particularly useful when it is 25 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), variations in single nucleotides between allelic forms of such nucleotide sequence, 30 can be used as perfect markers or candidate genes for the given trait.

11 Applicants have identified a number of SNPs of the nucleic acids and 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 sequences of nucleic acid fragments contributing to consensus contig 5 sequences. See for example, Figures 3, 6, 12, 15, 20, 23, 33, 46, 60, 67, 70, 73, 76, 79 and 85 (Sequence ID Nos. 3-7, 10-12, 19-21, 24-26, 31-37, 40-58, 69-77, 93-95, 111-115, 122-137, 140-144, 147-151, 154-156, 159-163, and 170-173, respectively). Accordingly, in a further aspect of the present invention, there is provided a 10 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 15 of isolating a nucleic acid or nucleic acid fragment of the present invention including a single nucleotide polymorphism (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. 20 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. 25 In a still further aspect of the present invention, there is provided use of nucleic acids or nucleic acid fragments of the present invention including SNPs, and/or nucleotide sequence information thereof, as molecular genetic markers.

12 In a still further aspect of the present invention there is provided use of a nucleic acid or nucleic acid fragment according to 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 5 present invention 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 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 10 used as molecular genetic markers in forage and turf grass improvement in relation to plant defense response, disease and/or pest resistance, protein breakdown, glucan and/or chitin breakdown, production of signals and elicitors for the manipulation of plant development, and/or host defense reaction, e.g. tagging QTLs for disease resistance, insect resistance, nematode resistance, leaf protein 15 breakdown, seed protein breakdown. 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 may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in ryegrasses and fescues. 20 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 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 25 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 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 30 vectors but should not be seen as being limited thereto.

13 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 5 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 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 10 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 15 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 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 20 chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant 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 25 and phage DNA. However, any other vector may be used as long as it is replicable, or 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.

14 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 those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible 5 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 promoter, and the rice Actin promoter. A variety of terminators which may be employed in the vectors of the 10 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 35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes. 15 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 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 20 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 pat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain a ribosome binding site for translation initiation. The 25 vector may also include 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, 30 histochemical GUS assays, northern and Western blot hybridisation analyses.

15 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 5 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 10 turfgrasses, corn, oat, sugarcane, wheat and barley), dicotyledons (such as 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) 15 and fescues (Festuca species), more preferably perennial ryegrass, 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 20 pratense), subterranean clover (Trifolium subterraneum) and lucerne (Medicago 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 25 transformation) are well known to those skilled in the art. Such techniques include 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 30 type of plant to be transformed.

16 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 5 skilled in the art. The resulting plants may be reproduced, either sexually or 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 or 10 vector 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 15 (Lolium species) or fescue (Festuca species), most preferably perennial 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 20 (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 25 derived from a plant of the present invention. In a further aspect of the present invention there is provided a method of modifying plant defense response, pest and/or disease resistance, protein breakdown, glucan or chitin breakdown, production of signals or elicitors for the manipulation of plant development, and/or host defense reaction, in a plant, said 17 method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or vector according to the present invention. By "an effective amount" it is meant an amount sufficient to result in an 5 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 suitable amount and method of administration. See, for example, Maniatis et al, Molecular 10 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, plant defense response, pest and/or disease resistance, protein breakdown, glucan and/or chitin breakdown, production of signals or elicitors for the manipulation of plant 15 development, or host defense reaction may be increased or decreased or otherwise modified relative to an untransformed control plant. For example, fungal disease resistance, viral disease resistance, bacterial disease resistance, insect pest resistance, nematode resistance, plant host response to pathogen challenge, or protein breakdown may be increased or otherwise altered. They may be 20 increased, for example, by incorporating additional copies of a sense nucleic acid or nucleic acid fragment of the present invention. They may be decreased, for 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 25 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.

18 In the Figures Figure 1 shows the consensus contig nucleotide sequence of LpPla (Sequence ID No. 1). Figure 2 shows the deduced amino acid sequence of LpPla (Sequence ID 5 No. 2). Figure 3 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPla (Sequence ID Nos. 3 to 7). Figure 4 shows the consensus contig nucleotide sequence of LpPlb 10 (Sequence ID No. 8). Figure 5 shows the deduced amino acid sequence of LpPlb (Sequence ID No. 9). Figure 6 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPlb (Sequence ID Nos. 10 15 to 12). Figure 7 shows the consensus contig nucleotide sequence of LpPIc (Sequence ID No. 13). Figure 8 shows the deduced amino acid sequence of LpPIc (Sequence ID No. 14). 20 Figure 9 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPIc (Sequence ID Nos. 15 and 16). Figure 10 shows the consensus contig nucleotide sequence of LpPId (Sequence ID No. 17).

19 Figure 11 shows the deduced amino acid sequence of LpPld (Sequence ID No. 18). Figure 12 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPld (Sequence ID Nos. 19 5 to 21). Figure 13 shows the consensus contig nucleotide sequence of LpPle (Sequence ID No. 22). Figure 14 shows the deduced amino acid sequence of LpPle (Sequence ID No. 23). 10 Figure 15 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPle (Sequence ID Nos. 24 to 26). Figure 16 shows the nucleotide sequence of LpPlf (Sequence ID No. 27). Figure 17 shows the deduced amino acid sequence of LpPlf (Sequence ID 15 No. 28). Figure 18 shows the consensus contig nucleotide sequence of LpPIg (Sequence ID No. 29). Figure 19 shows the deduced amino acid sequence of LpPIg (Sequence ID No. 30). 20 Figure 20 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPIg (Sequence ID Nos. 31 to 37). Figure 21 shows the consensus contig nucleotide sequence of LpPlh (Sequence ID No. 38).

20 Figure 22 shows the deduced amino acid sequence of LpPlh (Sequence ID No. 39). Figure 23 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPlh (Sequence ID Nos. 40 5 to 58). Figure 24 shows the consensus contig nucleotide sequence of LpPli (Sequence ID No. 59). Figure 25 shows the deduced amino acid sequence of LpPli (Sequence ID No. 60). 10 Figure 26 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPli (Sequence ID Nos. 61 and 62). Figure 27 shows the nucleotide sequence of LpPIj (Sequence ID No. 63). Figure 28 shows the deduced amino acid sequence of LpPlj (Sequence ID 15 No. 64). Figure 29 shows the nucleotide sequence of LpGLUCa (Sequence ID No. 65). Figure 30 shows the deduced amino acid sequence of LpGLUCa (Sequence ID No. 66). 20 Figure 31 shows the consensus contig nucleotide sequence of LpGLUCb (Sequence ID No. 67). Figure 32 shows the deduced amino acid sequence of LpGLUCb (Sequence ID No. 68).

21 Figure 33 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpGLUCb (Sequence ID Nos. 69 to 77). Figure 34 shows the nucleotide sequence of LpGLUCc (Sequence ID No. 5 78). Figure 35 shows the deduced amino acid sequence of LpGLUCc (Sequence ID No. 79). Figure 36 shows the nucleotide sequence of LpGLUCd (Sequence ID No. 80). 10 Figure 37 shows the deduced amino acid sequence of LpGLUCd (Sequence ID No. 81). Figure 38 shows the consensus contig nucleotide sequence of LpGLUCe (Sequence ID No. 82). Figure 39 shows the deduced amino acid sequence of LpGLUCe 15 (Sequence ID No. 83). Figure 40 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpGLUCe (Sequence ID Nos. 84 and 85). Figure 41 shows the consensus contig nucleotide sequence of LpGLUCf 20 (Sequence ID No. 86). Figure 42 shows the deduced amino acid sequence of LpGLUCf (Sequence ID No. 87). Figure 43 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpGLUCf (Sequence ID 25 Nos. 88 to 90).

22 Figure 44 shows the consensus contig nucleotide sequence of LpGLUCg (Sequence ID No. 91). Figure 45 shows the deduced amino acid sequence of LpGLUCg (Sequence ID No. 92). 5 Figure 46 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpGLUCg (Sequence ID Nos. 93 to 95). Figure 47 shows the nucleotide sequence of LpGLUCh (Sequence ID No. 96). 10 Figure 48 shows the deduced amino acid sequence of LpGLUCh (Sequence ID No. 97). Figure 49 shows the nucleotide sequence of LpGLUCi (Sequence ID No. 98). Figure 50 shows the deduced amino acid sequence of LpGLUCi (Sequence 15 ID No. 99). Figure 51 shows the nucleotide sequence of LpGLUCj (Sequence ID No. 100). Figure 52 shows the deduced amino acid sequence of LpGLUCj (Sequence ID No. 101). 20 Figure 53 shows the consensus contig nucleotide sequence of LpGLUCk (Sequence ID No. 102). Figure 54 shows the deduced amino acid sequence of LpGLUCk (Sequence ID No. 103).

23 Figure 55 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpGLUCk (Sequence ID Nos. 104 to 106). Figure 56 shows the nucleotide sequence of LpGLUCI (Sequence ID No. 5 107). Figure 57 shows the deduced amino acid sequence of LpGLUCI (Sequence ID No. 108). Figure 58 shows the consensus contig nucleotide sequence of LpGLUCm (Sequence ID No. 109). 10 Figure 59 shows the deduced amino acid sequence of LpGLUCm (Sequence ID No. 110). Figure 60 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpGLUCm (Sequence ID Nos. 111 to 115). 15 Figure 61 shows the nucleotide sequence of LpGLUCn (Sequence ID Nos. 116). Figure 62 shows the deduced amino acid sequence of LpGLUCn (Sequence ID No. 117). Figure 63 shows the nucleotide sequence of LpGLUCo (Sequence ID No. 20 118). Figure 64 shows the deduced amino acid sequence of LpGLUCo (Sequence ID No. 119). Figure 65 shows the consensus contig nucleotide sequence of LpCHITa (Sequence ID No. 120).

24 Figure 66 shows the deduced amino acid sequence of LpCHITa (Sequence ID No. 121). Figure 67 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCHITa (Sequence ID Nos. 5 122 to 137). Figure 68 shows the consensus contig nucleotide sequence of LpCHITb (Sequence ID No. 138). Figure 69 shows the deduced amino acid sequence of LpCHITb (Sequence ID No. 139). 10 Figure 70 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCHITb (Sequence ID Nos. 140 to 144). Figure 71 shows the consensus contig nucleotide sequence of LpCHITc (Sequence ID No. 145). 15 Figure 72 shows the deduced amino acid sequence of LpCHITc (Sequence ID No. 146). Figure 73 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCHITc (Sequence ID Nos. 147 to 151). 20 Figure 74 shows the consensus contig nucleotide sequence of LpCHITd (Sequence ID No. 152). Figure 75 shows the deduced amino acid sequence of LpCHITd (Sequence ID No. 153).

25 Figure 76 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCHITd (Sequence ID Nos. 154 to 156). Figure 77 shows the consensus contig nucleotide sequence of LpCHITe 5 (Sequence ID No. 157). Figure 78 shows the deduced amino acid sequence of LpCHITe (Sequence ID No. 158). Figure 79 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCHITe (Sequence ID Nos. 10 159 to 163). Figure 80 shows the consensus contig nucleotide sequence of LpCHITf (Sequence ID No. 164). Figure 81 shows the deduced amino acid sequence of LpCHITf (Sequence ID No. 165). 15 Figure 82 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCHITf (Sequence ID Nos. 166 and 167). Figure 83 shows the consensus contig nucleotide sequence of LpCHITg (Sequence ID No. 168). 20 Figure 84 shows the deduced amino acid sequence of LpCHITg (Sequence ID No. 169). Figure 85 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCHITg (Sequence ID Nos. 170 to 173).

26 Figure 86 shows the nucleotide sequence of LpCHITh (Sequence ID No. 174). Figure 87 shows the deduced amino acid sequence of LpCHITh (Sequence ID No. 175). 5 EXAMPLE Preparation of cDNA libraries, isolation and sequencing of cDNAs coding for PI and Pl-like, GLUC and GLUC-like and CHIT and CHIT-like proteins from perennial ryegrass (Lo/ium perenne) 10 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). TABLE 1 15 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) 1 Org 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 27 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) 21rg 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) 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. 15 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 28 provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut pBluescript 11 SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into E. coli DH10B cells according to 5 the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared 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 10 DNA preparation may be performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocol provided by Qiagen. Amplified insert DNAs are sequenced in dye-terminator sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"). The resulting ESTs are analyzed using an Applied Biosystems ABI 3700 sequence analyser. 15 EXAMPLE 2 DNA sequence analyses The cDNA clones encoding PI and PI-like, GLUC and GLUC-like and CHIT and CHIT-like proteins were identified by conducting BLAST (Basic Local Alignment 20 Search Tool; Altschul et a. (1993) 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 (NCBI). The DNA sequences were translated in all reading frames and compared 25 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 30 BLASTN algorithm. The identical and/or overlapping sequences were subjected to a multiple alignment using the CLUSTALw algorithm, and to generate a 29 consensus contig sequence derived from this multiple sequence alignment. The 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. 5 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 variants) as used in this specification is equivalent to the term "includes" and 10 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.

Claims

1. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding a proteinase inhibitor (PI) protein and including a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 20, 21, 23, 24, 26 and 27 hereto (Sequence ID Nos. 1, 3-7, 8,

10-12, 13, 15-16, 17, 19-21,22,24-26,27, 29, 31-37, 38,40-58, 59,61-62 and 63, 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). 2. A nucleic acid or nucleic acid fragment according to claim 1 wherein said functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequences recited in (a), (b) and (c) and have a size of at least 60 nucleotides. 3. A nucleic acid or nucleic acid fragment according to claim 1 wherein said functionally active fragment or variant includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 20, 21, 23, 24, 26 and 27 hereto (Sequence ID Nos. 1, 3-7, 8, 10 12, 13, 15-16, 17, 19-21, 22, 24-26, 27, 29, 31-37, 38, 40-58, 59, 61-62 and 63, respectively). 4. A nucleic acid or nucleic acid fragment according to any one of claims 1 to 3 wherein said nucleic acid or nucleic acid fragment is from a Lolium species. 5. A construct including a nucleic acid or nucleic acid fragment according to any one of Claims 1 to 4. 6. A vector including a nucleic acid or nucleic acid fragment according to any one of Claims 1 to 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. 31 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. 9. A plant, plant seed or other plant part derived from a plant cell or plant according to Claim 8 and including a construct according to claim 5 or a vector according to Claim 6 or 7. 10. A method of modifying plant defense response, pest and/or disease resistance, protein breakdown, glucan or chitin breakdown, production of signals or elicitors for the manipulation of plant development, and/or host defense reaction, 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 to 4, a construct according to Claim 5 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 to 4, and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof as a molecular genetic marker.

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

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

14. A substantially purified or isolated PI polypeptide, said polypeptide including an amino acid sequence selected from the group consisting of sequences shown in Figures 2, 5, 8, 11, 14, 17, 19, 22, 25 and 28 hereto (Sequence 10 Nos. 2, 9, 14, 18, 23, 28, 30, 39, 60 and 64, respectively); and functionally active fragments and variants thereof.

15. A polypeptide according to claim 14 wherein said functionally active fragments and variants have at least approximately 90% identity with the recited sequences and have a size of at least 20 amino acids. 32

16. A polypeptide according to claim 14 wherein said polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 2, 5, 8, 11, 14, 17, 19, 22, 25 and 28 hereto (Sequence 10 Nos. 2, 9, 14, 18, 23, 28, 30, 39, 60 and 64, respectively).

17. A polypeptide according to any one of claims 14 to 16 wherein said polypeptide is from a Lolium species.

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

19. A polypeptide according to claim 14 substantially as hereinbefore described with reference to any one of the Figures or Examples.