WO1999045118A1

WO1999045118A1 - Genetic sequences conferring pathogen resistance in plants and uses therefor

Info

Publication number: WO1999045118A1
Application number: PCT/AU1999/000130
Authority: WO
Inventors: Anthony John Pryor; Jeffrey Graham Ellis; Nicholas Charles Collins; Scot Hulbert
Original assignee: Commonwealth Scientific And Industrial Research Organisation; Kansas State University Research Foundation; Grains Research And Development Corporation
Priority date: 1998-03-06
Filing date: 1999-03-03
Publication date: 1999-09-10
Also published as: AU2820099A; CA2322719A1; ZA991794B; EP1062342A1; AR014683A1

Abstract

The present invention provides isolated genetic sequences derived from barley and maize that confer, or otherwise facilitate or enhance, resistance in plants to plant pathogens, in particular, against Puccinia sorghi and barley stem rust, and genetic constructs comprising said genetic sequences. The present invention further provides for plants into which the subject genetic sequences have been introduced, generating enhanced resistance qualities to plant fungal pathogens and for methods of producing same.

Description

- 1 -

GENETIC SEQUENCES CONFERRING PATHOGEN RESISTANCE IN PLANTS AND USES THEREFOR

FIELD OF THE INVENTION The present invention relates generally to isolated genetic sequences derived from plants and more particularly to isolated genetic sequences derived from plants which confer, or otherwise facilitate or enhance, resistance in plants to plant fungal pathogens, such as fungi, rusts, nematodes, viruses and bacterial pathogens. The present invention further provides for plants into which the subject genetic sequences have been introduced, generating enhanced resistance qualities to plant fungal pathogens. The present invention is particularly useful in the development of plants which have improved resistance to plant fungal pathogens, in particular cereal crop plants which have improved resistance to rusts.

GENERAL

Bibliographic details of the publications referred to in this specification are collected at the end of the description.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular specified source or species, albeit not necessarily directly from that specified source or species.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or - 2 -

features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Sequence identity numbers (SEQ ID NOS.) containing nucleotide and amino acid sequence information included in this specification are collected after the Abstract and have been prepared using the programme Patentin Version 2.0. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1 , <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400>1, <400>2, etc).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

The designation of amino acid residues referred to herein, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission, are listed in Table 1. TABLE 1

Amino Acid Three-letter code One-letter code

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Cysteine Cys C Glutamine Gin Q

Glutamic acid Glu E

Glycine Gly G

Histidine His H

Isoleucine Ile I Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P Serine Ser S

Threonine Thr T

Tryptophan Trp w

Tyrosine Tyr Y

Valine Val V Aspartate/Asparagine Baa B

Glutamate/Glutamine Zaa z

Any amino acid Xaa X

BACKGROUND TO THE INVENTION

Improvements in recombinant DNA technology have produced dramatic changes to the agricultural industry, in particular the approaches taken to improve crop productivity. A major concern is the effect of plant pests, such as plant fungal pathogens, on productivity. Plant pathogens such as fungi, rusts, nematodes, viruses and bacteria invade a wide range of food, fibre and ornamental plants, causing damage to different plant tissues reducing productivity.

Plant fungal pathogens, in particular rust fungi, represent an especially significant problem amongst broadacre crops such as legume and cereal grains. Biotechnology offers considerable scope for addressing this problem, by introducing recombinant genes into plants that either kill or disable a fungal pathogen, or restrict a fungal pathogen to a limited zone of infection, thereby preventing significant deterioration of an economically-important crop.

An interaction between a rust pathogen and a plant host may be classed as either "resistant" or "susceptible" depending on how the fungal infection proceeds. In a resistant interaction, infection by a fungal pathogen produces a "plant hypersensitive response" (Marineau etal., 1987; Dixon and Lamb, 1990) resulting in cell death to limit spread of the fungus. During the hypersensitive response, the expression of several infection-related genes, for example genes encoding phytoalexins, antimicrobial agents and pathogenesis-related (PR) proteins, is switched on. In contrast, a susceptible interaction involves no hypersensitive cell death and the infection alters host cell gene expression in such a way as to provide gene products that are essential for the biotrophic growth of an obligate plant pathogen. Additionally, hos pathogen interactions may be partially susceptible, in which case there may be a delay of several days in the appearance of PR proteins and the level of their expression is reduced compared to fully hypersensitive responses. It is also possible that both resistant and susceptible interactions may operate to varying degrees in any given pathogenic infection. - 5 -

Although several studies have concentrated on characterising hos pathogen interactions during fungal infection of plants at the genetic level, in particular in a resistant interaction between the host plant and fungal pathogen, the identification of host-derived genes capable of conferring pathogen-resistance on plants has not been a straightforward procedure. In particular, although fungal pathogens are a major infections agent of monocotyledonous broadacre crop plants, the generation time and genome organisation of these plants has rendered the isolation of such sequences difficult, even in light of well-characterised genetic systems. Moreover, those skilled in the art would be aware of the difficulties associated with the extrapolation of mere genetic linkage data to physical distances between loci in the complex genome of a monocotyledonous plant.

SUMMARY OF THE INVENTION

In accordance with the present invention, genetic sequences conferring resistance to a plant pathogen, such as a plant fungal, nematode, viral or bacterial pathogen, have been cloned from Zea mays and Hordeum spp. The cloning of these sequences permits the generation of transgenic plants with de novo, improved or otherwise enhanced pathogen resistance, in particular resistance against fungal pathogens, such as rusts, and more particularly, rusts of the genera Puccinia or related rusts capable of infecting cereal crop plants.

The nucleotide sequences exemplified herein provide the means by which variant gene sequences may be isolated which confer resistance to a wide range of plant pathogens such as viruses, fungi, bacteria and nematodes and more particularly wheat streak mosaic virus, barley stem rust, Pyricularia ssp. and Xanthomonas ssp.

The present invention also permits the screening through genetic or immunological means, similar pathogen resistance genes in other plants for use in developing or enhancing pathogen resistance in commercially and economically important species.

Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising an Rp1-D nucleotide sequence or Rpg1 nucleotide sequence or a variant thereof which encodes a protein or derivative thereof, which confers, enhances, or otherwise facilitates resistance to a pathogen in a plant.

In another embodiment, the present invention provides an isolated nucleic acid molecule derived from a plant and comprising an Rp1-D nucleotide sequence or an Rpg1 nucleotide sequence or a variant thereof which:

(i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates pathogen resistance in a plant; and

(ii) has at least about 60% nucleotide sequence identity to any one or more of the nucleotide sequences set forth in SEQ ID NOS: <400>1 or <400>3 or <400>5 or <400>7, or SEQ ID Nos:<400>64 to <400>70, or a part thereof.

In yet another embodiment, the present invention provides an isolated nucleic acid molecule derived from a plant and comprising an Rp1-D nucleotide sequence or an Rpg1 nucleotide sequence or a variant thereof which:

(ii) hybridises under at least low stringency conditions to the Rp1-D nucleotide sequence set forth in SEQ ID NOS: <400>1 or <400>3 or <400>5 or <400>7 or SEQ ID Nos:<400>64 to <400>70 or to a complementary strand thereof.

In yet another embodiment, the invention provides an isolated nucleic acid molecule which is substantially the same as any one or more of the Rp1-D nucleotide sequences set forth in SEQ ID NOS: <400>1 or <400>3 or the Rpg1 nucleotide sequences set forth in SEQ ID NOS:<400>5 or <400>7, or the hrpl sequences set forth in SEQ ID Nos:<400>64 to <400>70 or which is at least about 60% identical thereto. Another aspect of the invention provides a genetic construct comprising an Rp1-D nucleotide sequence or an Rpg1 nucleotide sequence or a variant thereof derived from a plant and encoding a protein or derivative thereof, which confers, enhances, or otherwise facilitates resistance to a pathogen in a plant or a complementary nucleotide sequence thereto. According to one embodiment, the Rp1-D nucleotide sequence or Rpg1 nucleotide sequence is operably linked to a promoter sequence, thereby regulating expression of said nucleotide sequence in a eukaryotic cell, for example a plant cell, or a prokaryotic cell.

The invention extends to the recombinant Rp1-D polypeptide product or Rpg1 polypeptide product of said genetic construct.

The present invention also provides an oligonucleotide molecule of at least 10 nucleotides in length capable of hybridising under low stringency conditions to part of the Rp1-D nucleotide sequence set forth in SEQ ID NOS: <400>1 or <400>3 or the Rpg1 nucleotide sequences set forth in SEQ ID NOS:<400>5 or <400>7, or the hrpl nucleotide sequences set forth in SEQ ID Nos:<400>64 to <400>70 or to a complementary nucleotide sequence thereto.

The Rp1-D nucleotide sequence and/or hrpl nucleotide sequence and/or Rpg1 nucleotide sequence and/or oligonucleotides comprising same or hybridising thereto are useful in the isolation of pathogen resistance or pathogen resistance-like genetic sequences from other plants, including those genetic sequences which are involved in resistance against both fungal pathogens and non-fungal pathogens, using hybridisation and/or PCR-based approaches.

Accordingly, there is provided a method of identifying a pathogen resistance genetic sequence or pathogen resistance-like genetic sequence which method comprises contacting genomic DNA, or mRNA, or cDNA, or parts, or fragments thereof, or a source thereof with an Rp1-D nucleotide sequence or with an Rpg1 nucleotide sequence or with an hrpl nucleotide sequence or a variant thereof which encodes a - 8 -

polypeptide which confers, enhances or otherwise facilitates pathogen resistance, or a part thereof, or a complementary nucleotide sequence thereto, and then detecting said hybridisation.

There is also provided a method of identifying a pathogen resistance genetic sequence or a pathogen resistance-like genetic sequence in a plant cell, which method comprises contacting genomic DNA, mRNA, or cDNA from said plant with one or more oligonucleotide molecules derived from an Rp1-D nucleotide sequence or an hrpl nucleotide sequence or an Rpg1 nucleotide sequence or a variant thereof which encodes a polypeptide which confers, enhances or otherwise facilitates pathogen resistance, or a part thereof, or a complementary nucleotide sequence thereto, for a period of time and under conditions sufficient to form a double-stranded nucleic acid molecule and amplifying copies of the said genetic sequence in a polymerase chain reaction.

In another aspect, this invention also provides an isolated Rp1-D polypeptide or an Rpg1 polypeptide or a functional mutant, derivative part, fragment, or analogue of said polypeptide which confers, enhances, or otherwise facilitates resistance to a pathogen in a plant cell.

The present invention extends to a synthetic peptide comprising at least 10 contiguous amino acids of the Rp1-D amino acid sequence set forth in SEQ ID NO: <400>2 or SEQ ID NO: <400>4 or the Rpg1 amino acid sequence set forth in SEQ ID NO: <400>6 or SEQ ID NO: <400>8 or having at least about 60% identity to all or a part thereof.

The polypeptide and synthetic peptides of the present invention may be used to generate specific immuno-interactive molecules. Accordingly, the present invention also contemplates an antibody that binds to an Rp1-D polypeptide or Rpg1 polypeptide which confers, enhances or otherwise facilitates resistance to a pathogen in a plant or an antigenic or immunogenic epitope thereof, wherein said polypeptide or epitope further comprises an amino acid sequence which is substantially the same as the amino acid sequence set forth in SEQ ID NO: <400>2 or SEQ ID NO: <400>4 or SEQ ID NO: <400>6 or SEQ ID NO: <400>8 or is at least about 60% identical to all or a part thereof.

In yet another aspect of the present invention, there is provided a method of identifying a pathogen resistance gene product or pathogen resistance-like gene product in a plant cell, which method comprises contacting an antibody which is specific for an Rp1- D polypeptide or an Rpg1 polypeptide or an antigenic fragment or epitope thereof with an antigen from said plant for a period of time and under conditions sufficient to form an antibody-antigen complex and measuring the amount of said antibody-antigen complex formed.

Notwithstanding that the genetic sequences described herein are derived from plants by virtue of their fungal-resistance properties, the genetic sequences of the present invention are also useful for the generation of plants with generally-enhanced pathogen resistance characteristics, in particular resistance against a fungal, nematodes, viral or bacterial pathogen. Accordingly, there is also provided a plant carrying a non- endogenous Rp1-D nucleotide sequence or a non-endogenous Rpg1 nucleotide sequence or a variant thereof which when expressed in said plant confers, enhances, or otherwise facilitates pathogen resistance in said plant. The present invention extends to the progeny derived from said plant.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphical representation showing the Rp1 region of maize chromosome 10S showing the linkage relationships of genes for resistance against rusts (Rp), the Rp1-D gene and several RFLP or molecular markers.

Figure 2 is a copy of a photographic representation of Southern blot hybridisation of Λ/col and Sg7ll (panel B) restriction digests of DNA isolated from maize lines carrying different alleles of the Rp locus including Rp1-D. The Rp alleles present in each lane - 10 -

θf panel A are as follows: Lane 1 , rp-R168; Lane 2, Rp1-A; Lane 3, Rp1-B; Lane 4, blank; Lane 5, Rp1-C; Lane 6, Rp1-D; Lane 7, Rp1-F; Lane 8, Rp1-J, Lane 9, Rp1-M; Lane 10, Rpf-/; Lane 11 , Rp1-K; Lane 12, Rp1-C-K; Lane 13, ftpf-G; Lane 14, Rp1-G- I, Lane 15, Rp1-G-5; Lane 16, Rp1-G-F-J; Lane 17, f?p5; Lane 18, Rpϊ-Td; Lane 19, Rp5-C; Lane 20, Rp5-M; Lane 21 , RpG-M; Lane 22, Rp1-D-13-2; Lane 23, Rp1-D13; Lane 24, rp-W22; Lane 25, rp-a_ts/7₂. The ftp 7 alleles present in each lane of panel B are as follows: Lane 1, rp-R168; Lane 2, Rp1-A; Lane 3, Rp7-B; Lane 4, Rp7-C; Lane 5, Rp7-D; Lane 6, Rp1-F; Lane 7, Rp1-J, Lane 8, Rp?-/W; Lane 9, Rp1-I; Lane 10, ftp7- ; Lane 11 , Rp1-C-K; Lane 12, Rp7-G; Lane 13, / ϊ-G-/, Lane 14, Rp1-G-5; Lane 15, Rp1-G-F-J Lane 16, Rp5; Lane 17, Rp1-Td; Lane 18, Rp5-C; Lane 19, Rp5-M; Lane 20, RpG-M; Lane 21 , Rp1-D-13-2; Lane 22, Rp1-D13; Lane 23, rp-lΛ/22; Lane 24, rp- a,s/ι₂. The filters were probed with the Rp1-D probe.

Figure 3 is a copy of a photographic representation of a Southern blot hybridisation of barley DNA isolated from individuals of a segregating family, digested with Oral and probed with the RpD-1 probe to show cross hybridisation and linkage with the Rpgλ rust-resistance locus in barley specifying resistance to barley rust, P. graminis. Rpg1

= rust resistance; rpgl = rust susceptible. Arrows indicate the position of DNA bands which co-segregate with rust resistance.

Figure 4A is a representation of an amino acid sequence alignment of the maize Rp1-

D and barley Rpg1-2 and barley Rpg1-13 polypeptides.

Figure 4B is a diagrammatic representation of the Rpg1-2 and Rp1-D polypeptides, illustrating the similarity in amino acid sequences thereof. Figure 4B-I shows the relative location of kinase-1a (p-loop) and kinase-2a and leucine-rich repeat (LRR) regions of these polypeptides. The overall percentages identity and similarity are indicated at the bottom of the Figure. Figure 4B- II shows the Kyte-Dolittle hydropathy plots of the Rpg1-2 polypeptide (top) and Rp1-D polypeptide (lower).

Figure 5 is a representation of a nucleotide sequence alignment between the coding - 11 -

regions of the maize Rp1-D gene and the barley Rpg1-2 and barley Rpg1-13 alleles.

Figure 6 is a copy of a photographic representation of a Southern blot hybridization of Λ/col (left) or Bg1\\ (right) digested DNA from various grass species detected using the probe from the f?p7-D gene. The Figure shows cross hybridisation of the Rp1-D gene sequence with sorghum (Sorghum vulgare; lane 1); pearl millet (Panicum mileraceum; lane 2); maize (Zea mays; lane 3); sugar cane (Saccharum officinale; lane 4); barley (Hordeum vulgare cv Morex; lane 5); wheat (Triticum aestivum cv Katyil; lane 6); oats (Avena sativa cv Marbo; lane 7); Triticum tauschii (lane 8); and rice (Oryza sativa cv Fuji; lane 9).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention comprises an isolated nucleic acid molecule comprising an Rp1-D nucleotide sequence or an Rpg1 nucleotide sequence or a variant thereof which encodes a polypeptide which confers, enhances or otherwise facilitates resistance to a pathogen in a plant or a complementary nucleotide sequence thereto.

As used herein, the term "Rp1-D nucleotide sequence" shall be taken to refer to a nucleotide sequence that is the same as the maize Rp1-D gene set forth herein, or a fragment comprising at least about 20-30 contiguous nucleotide residues thereof or a complementary nucleotide sequence thereto.

As used herein, the term "Rpg1 nucleotide sequence" shall be taken to refer to a nucleotide sequence that is the same as the barley Rpg1-2 and/or barley Rpg1-13 alleles set forth herein, or a fragment comprising at least about 20-30 contiguous nucleotide residues thereof or a complementary nucleotide sequence thereto.

Hereinafter the term "pathogen resistance gene" or "pathogen resistance-like gene", or similar term shall be used to define a nucleic acid molecule which upon expression confers, enhances, or otherwise facilitates resistance of a cell and/or organism to one - 12 -

or more plant pathogens. The term "pathogen resistance gene" further defines a nucleic acid molecule which upon expression confers, enhances, or otherwise facilitates resistance to one or more plant pathogens selected from the list comprising fungal pathogens such as rusts, viral pathogens, bacterial pathogens and parasites such as nematodes. Particularly preferred pathogen resistance genes are those which are capable of conferring resistance against fungal or viral pathogens wherein said gene is a variant of the maize Rp1-D nucleotide sequence or a variant of the barley Rpg1 nucleotide sequences exemplified herein.

As used herein, the term "variant of an Rp1-D nucleotide sequence" or "Rp1-D variant" or similar term shall be taken to refer to any pathogen resistance gene derived from a plant wherein said gene comprises a nucleotide sequence which is at least about 60% identical to the Zea mays Rp1-D gene exemplified herein including the hrpl nucleotide sequences set forth as SEQ ID Nos:<400>64 to <400>70 exemplified herein; or a fragment thereof comprising at least about 30 nucleotides in length; or a pathogen resistance gene or fragment that hybridises to the Zea mays Rp1-D nucleotide sequence gene exemplified herein, or to a complementary sequence thereto, under at least low stringency hybridisation conditions. An Rp1-D variant may include an allelic variant of the specific Rp1-D gene sequence exemplified herein, in which case said allelic variant exhibits similar biological activity (i.e. against a similar or related pathogen). Alternatively, an Rp7-D variant may be derived from Zea mays or another plant species and exhibit different pathogen resistance characteristics to the Zea mays Rp1-D gene exemplified herein, for example a functionally-distinct Rp1 allele or other related Rp allele.

Similarly, the term "variant of an Rpg1 nucleotide sequence" or "Rpg1 variant" or similar term shall be taken to refer to any pathogen resistance gene derived from a plant wherein said gene comprises a nucleotide sequence which is at least about 60% identical to the barley Rpg1-2 or barley Rpg1-13 alleles exemplified herein; or a fragment thereof comprising at least about 30 nucleotides in length; or a pathogen resistance gene or fragment that hybridises to the barley Rpg1-12 or barley Rpg1-13 - 13 -

alleles exemplified herein, or to a complementary sequence thereto, under at least low stringency hybridisation conditions. An Rpg1 variant may include an allelic variant of the specific Rpg1-2 and/or Rpg1-13 allelic gene sequences exemplified herein, in which case said allelic variant exhibits similar biological activity (i.e. against a similar or related pathogen). Alternatively, an Rpg1 variant may be derived from Hordeum spp. or another plant species and exhibit different pathogen resistance characteristics to the Hordeum spp. Rpg1-2 and Rpg1-13 alleles exemplified herein, for example a functionally-distinct Rpg1 allele or other related Rpg1 allele.

The term "Rp1-D variant polypeptide" or similar shall be taken to refer to the polypeptide product of an Rp1-D variant gene as hereinbefore defined, in particular a polypeptide produced of the hrpl nucleotide sequence set forth in any one or more of SEQ ID Nos:<400>64 to <400>70, or an immunological or functional homologue, analogue or derivative thereof.

The term "Rpg1 variant polypeptide" or similar shall be taken to refer to the polypeptide product of an Rpg1 variant gene as hereinbefore defined or an immunological or functional homologue, analogue or derivative thereof.

Preferably, Rp1-D and Rpg1 variants are isolated from broadacre crop plants and more preferably from monocotyledonous broadacre crop plants such as cereals and grasses, for example Zea mays, pearl millet, sugar cane, wheat, barley, rye, rice, oats, triticale, sorghum and ryegrass.

In particular, the present inventors have isolated nucleic acid molecules comprising one or more Rp7-D and/or hrpl alleles and/or Rpg1 alleles which confer resistance to the rusts Puccinia sorghi (Rp1-D) and barley stem rust (Rpg1). The maize Rp1-D allele has been isolated from transposon-tagged lines of Zea mays. The isolated Rp1-D allele exemplified herein has been used as a hybridisation probe, to isolate the maize hrpl nucleotide sequences and barley Rpg1 nucleotide sequences exemplified herein. Accordingly, the maize hrp1-d1, hrp1-d2, hrp1-d3, hrp1-d4, hrp1-d5 and hrp1-d6 and - 14 -

hrp1-cin4 nucleotide sequences and the barley Rpg1-2 and Rpg1-13 nucleotide sequences exemplified herein fall within the scope of the term "Rp1-d variant" as defined herein. Similarly, the maize f?p7-D nucleotide sequence and maize hrp1-d1 to hrp1-d6 and hrp1-cin4 sequences exemplified herein is an "Rpg1 variant" as defined herein.

The inventors have also demonstrated the utility of the subject Rp1-D allele for the identification and/or characterisation of Rp1-D variants derived from other plant species, including allelic and non-allelic variants of the Zea mays Rp1-D nucleotide sequence in maize and other species.

Accordingly, particularly preferred Rp1-D variants contemplated herein include nucleotide sequences of the barley Rpg1 gene family and other alleles of the Rp1 gene family derived from a broadacre crop plant selected from the list comprising barley, pearl millet, maize, sugar case, wheat, oats, Triticum tauschii and rice.

In an even more particularly preferred embodiment, the pathogen-resistance gene of the present invention further comprises a sequence of nucleotides which is at least about 60% identical to at least about 30 contiguous nucleotides of the Zea mays Rp1-D nucleotide sequence set forth in SEQ ID NOS: <400>1 or <400>3 or the Zea mays hrpl nucleotide sequences set forth in SEQ ID NOS:<400>64 to <400>70 or a complementary sequence thereto, or the Hordeum spp. Rpg1 nucleotide sequences set forth in SEQ ID NOS: <400>5 or <400>7 or a complementary sequence thereto.

For the purposes of nomenclature, the sequences shown in SEQ ID NOS: <400>1 and <400>3 relate to a Rp1-D resistance allele derived from Zea mays which controls resistance to the rust pathogen Puccinia sorghi in a resistant interaction. More particularly, SEQ ID NO: <400>1 describes a genomic clone isolated from Zea mays, containing the Rp1-D allele 13. The nucleotide sequence set forth in SEQ ID NO: <400>3 is an Rp1-D cDNA clone which was isolated from Zea mays. - 15 -

The amino acid sequence encoded by the genomic Rp7-D gene is set forth in SEQ ID NO: <400>2. The amino acid sequence of the complete Rp1-D polypeptide encoded by the Rp1-D cDNA sequence is presented in SEQ ID NO: <400>4.

The nucleotide sequences shown in SEQ ID NOS: <400>5 and <400>7 relate to the Rpg1-2 and Rpg1-13 resistance alleles derived from Hordeum spp. which controls resistance to barley stem rust in a resistant interaction. More particularly, SEQ ID NO: <400>5 describes a genomic clone isolated from barley, containing an approximately 8.2 kb EcoRI-to-Sa/l fragment of the Rpg1-2 allele. The nucleotide sequence set forth in SEQ ID NO: <400>7 corresponds to the barley Rpg1-13 allele.

The amino acid sequence encoded by the genomic Rpg1-2 gene is set forth in SEQ ID NO: <400>6. The amino acid sequence of the Rpg1-13 polypeptide encoded by the Rpg1-13 allele is presented in SEQ ID NO: <400>8.

The present invention clearly extends to any one or more of the above-mentioned isolated nucleic acids or functional fragments, homologues, analogue or derivatives thereof, when integrated into a plant genome and to propagated plants containing one or more of said nucleic acid molecules, fragments, homologues, analogues or derivatives.

The f?p7-D variant or Rpg1 variant of the invention may confer resistance to a diverse range of fungal pathogens including stem rusts, leaf rust, yellow rust and crown rust, or to one or more nematode, viral or bacterial pathogens, amongst others. In a particularly preferred embodiment, the Rp1-D variant or Rpg1 variant of the present invention is a rust-resistance gene or a fragment or derivative thereof. More preferably, the Rp1-D variant or Rpg1 variant of the present invention is capable of conferring resistance against Puccinia and/or against barley stem rust in a plant.

Reference herein to a "gene" is to be taken in its broadest context and includes:

(i) a classical genomic gene consisting of a coding region optionally - 16 -

together with transcriptional and/or translational regulatory sequences and a coding region with or without non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or

(ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and optionally 5'- and 3'- untranslated sequences of the gene.

The term "gene" is also used to describe a synthetic or fusion molecule, or derivative which encodes, or is complementary to a molecule which encodes, all or part of a functional product. A functional product is one which confers, enhances or otherwise facilitates resistance of a cell to a fungal pathogen.

Genetic analysis indicates that specific interactions may occur between resistance genes and gene products of the pathogen. Although not intending to limit the present invention to any one theory or mode of action, it is proposed that the genetic sequences of the present invention may control host range via specific recognition of the gene products of the pathogen pest, in a "gene-for-gene" interaction that is understood by one normally skilled in the art. Accordingly, the genetic sequences are useful in increasing the range of resistance of plants to distinct pathogen pests, by providing de novo the required pathogen resistance gene, or being introduced together with the corresponding pathogen gene or genes, on, for example, a single genetic cassette. Accordingly, these aspects of the invention are covered by the expression "conferring, improving, or otherwise enhancing pathogen resistance" or other similar expression.

The present invention clearly extends to any Rp1-D variant or Rpg1 variant which comprises a pathogen resistance gene and any functional gene, mutant, derivative, part, fragment, homologue or analogue thereof or non-functional molecule which is at least useful as, for example, a genetic probe, or primer sequence in the enzymatic or chemical synthesis of said gene, or in the generation of immunologically interactive recombinant molecules. - 17 -

ln a particularly preferred embodiment, the Rp1-D nucleotide sequences and/or the Rpg1 nucleotide sequences of the present invention (or like genetic sequences) are employed to identify and isolate similar genes, or pathogen resistance-like genes from other plants. The present invention extends to the use of said genetic sequence, or a part thereof to detect polymorphisms of a pathogen resistance genetic sequence or pathogen resistance-like genetic sequence.

Allelic and other variants of the Rp1-D nucleotide sequences and/or hrpl nucleotide sequences and/or Rpg1 nucleotide sequences may be detected and/or isolated using the Rp7-D and/or Rpg1 nucleotide sequences exemplified herein by any means known to those skilled in the art. In particular, the nucleotide sequences disclosed herein provide the means for isolation and/or detection of related nucleotide sequences, for example other Rp1 alleles and Rpg1 alleles, using standard nucleic acid hybridisation or amplification approaches, without undue experimentation.

Those skilled in the art will be aware that a functional assignment of an Rp1-D variant and/or an Rpg1 variant identified by hybridisation and/or amplification approaches may further require genetic linkage or cosegregation data to demonstrate that said allele is closely associated with a particular resistance phenotype. Those skilled in the art will be readily capable of performing such embodiments without undue experimentation.

Accordingly, in a further aspect of the invention, there is provided an oligonucleotide molecule of at least about 10 nucleotides in length and more preferably at least about 25-30 nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of any one or more of the nucleotide sequences set forth in SEQ ID NOS: <400>1 and/or <400>3 and/or <400>5 and/or <400>7 and/or <400>64 to <400>70.

A further aspect of the present invention contemplates an isolated nucleic acid molecule which encodes a protein that confers or otherwise facilitates pathogen resistance in a plant and which is capable of hybridising under at least low stringency - 18 -

conditions to the nucleic acid molecule set forth in any one or more of SEQ ID NOS: <400>1 and/or <400>3 and/or <400>5 and/or <400>7 and/or any one of SEQ ID NOS:<400>64 to <400>70 or to a derivative, homologue or analogue thereof. Preferably, said nucleic acid molecule is an Rp1-D variant or an Rpg1 variant.

For the purposes of defining the level of stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in δxSSC buffer, 0.1% (w/v) SDS at 28 °C. Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of parameters affecting hybridisation between nucleic acid molecules, reference can conveniently be made to pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.

Preferably, variants of the Rp1-D and/or Rpg1 alleles exemplified herein are isolated by hybridisation under medium or more preferably, under high stringency conditions, to a probe which comprises at least about 30 contiguous nucleotides derived from SEQ ID NOS: <400>1 and/or <400>3 and/or <400>5 and/or <400>7 and/or any one of SEQ ID NOS:<400>64 to <400>70 or a complement thereof.

The Rp1-D and/or Rpg1 variant gene sequences thus isolated may be modified by standard recombinant techniques. Generally, a pathogen resistance gene may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions. Nucleotide insertional derivatives of the pathogen resistance gene of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more nucleotides from the sequence. - 19 -

Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place. Such a substitution may be "silent" in that the substitution does not change the amino acid defined by the codon. Alternatively, substituents are designed to alter one amino acid for another similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity.

Another aspect of the present invention is directed to a nucleic acid molecule which comprises a sequence of nucleotides corresponding or complementary to the nucleotide sequence set forth in any one of SEQ ID NOS: <400>1 or <400>3 or <400>5 or <400>7 and/or SEQ ID NOS:<400>64 to <400>70 or a homologue, analogue or derivative thereof having at least about 60% identity thereto, wherein said nucleic acid molecule encodes a protein which confers, enhances, or otherwise facilitates resistance to a pathogen in a plant.

Preferably, the percentage similarity to any one of said sequences set forth is at least about 70%. Even more preferably, the percentage similarity is at least 80-90%, including at least 91% or 93% or 95%.

In determining whether or not two nucleotide sequences fall within these percentage limits, those skilled in the art will be aware that it is necessary to conduct a side-by-side comparison or multiple alignment of sequences. In such comparisons or alignments, differences may arise in the positioning of non-identical residues, depending upon the algorithm used to perform the alignment. In the present context, reference to a percentage identity between two or more nucleotide sequences shall be taken to refer to the number of identical residues between said sequences as determined using any standard algorithm known to those skilled in the art. For example, nucleotide sequences may be aligned and their identity calculated using the BESTFIT programme or other appropriate programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, 1984). Alternatively or in addition, wherein two or more nucleotide sequences are - 20 -

being compared, the ClustalW programme of Thompson et al (1994) is used, as is the case for data presented herein as Figure 5.

For the present purpose, "homologues" of a nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

"Analogues" of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

"Derivatives" of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence identity to said sequence or a part thereof. Generally, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5^' and 3^' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues. Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed. Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue - 21 -

inserted in its place.

Particularly preferred homologues, analogues or derivatives of the nucleotide sequences of the present invention include any one or more of the isolated nucleic acid molecules selected from the following:

(i) an isolated nucleic acid molecule which comprises a nucleotide sequence which is at least about 60% identical to any one of SEQ ID NOS: <400>1 and/or <400>3 and/or <400>5 and/or <400>7 and/or SEQ ID NOS:<400>64 to <400>70 or a complementary sequence thereto; (ii) an isolated nucleic acid molecule which comprises a nucleotide sequence which is at least about 60% identical to at least about 30 contiguous nucleotides of SEQ ID NOS: <400>1 and/or <400>3 and/or <400>5 and/or <400>7 or any one of SEQ ID NOS:<400>64 to <400>70 or a complementary sequence thereto; (iii) an isolated nucleic acid molecule which is capable of hybridising under at least low stringency conditions to at least 10 contiguous nucleotides, preferably at least about 25-30 contiguous nucleotides of SEQ ID NOS: <400>1 and/or <400>3 and/or <400>5 and/or <400>7 or any one of SEQ ID NOS:<400>64 to <400>70 or a complementary sequence thereto; (iv) an isolated nucleic acid molecule which comprises a sequence of nucleotides which encodes or is complementary to a sequence of nucleotides which encodes an Rp1-D variant polypeptide;

(v) an isolated nucleic acid molecule which comprises a sequence of nucleotides which encodes or is complementary to a sequence of nucleotides which encodes an Rpg1 variant polypeptide

(vi) an isolated nucleic acid molecule which comprises a nucleotide sequence or is complementary to a nucleotide sequence which encodes a pathogen-resistance polypeptide which is at least about 60% identical at the amino acid sequence level to the amino acid sequence set forth in SEQ ID NOS: <400>2 and/or <400>4 and/or <400>6 and/or <400>8; and

(vii) an isolated nucleic acid molecule which comprises a nucleotide - 22 -

sequence or is complementary to a nucleotide sequence which encodes a pathogen-resistance polypeptide which at least comprises about 10 contiguous amino acids of the sequence set forth in SEQ ID NOS: <400>2 and/or <400>4 and/or <400>6 and/or <400>8.

A further aspect of the invention contemplates a method for identifying a pathogen resistance genetic sequence or pathogen resistance-like genetic sequence, said method comprising contacting genomic DNA, or mRNA, or cDNA, or parts, or fragments thereof, or a source thereof, with a hybridisation effective amount of a probe comprising an Rp1-D nucleotide sequence or an hrpl nucleotide sequence or an Rpg1 nucleotide sequence or a related pathogen resistance gene or a homologue, analogue or derivative thereof for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation.

The pathogen resistance genetic sequence or like sequence may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, said genetic sequence originates from a broadacre crop plant, in particular a monocotyledonous plant such as maize, barley, rye, oats, wheat, sorghum, triticale, ryegrass or rice and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same. In addition, the identified pathogen resistance genetic sequence or the probe may be bound to a support matrix, for example nylon, nitrocellulose, polyacrylamide, agarose, amongst others.

Preferably, the probe comprises the nucleotide sequence of the Zea mays Rp1-D variant exemplified herein or the barley Rpg1 variant allele exemplified herein or a related sequence such as, but not limited to, an Rp7-D nucleotide sequence or an hrpl nucleotide sequence or an Rpg nucleotide sequence derived from another plant species, amongst others.

In a most preferred embodiment, the probe comprises the sequence of nucleotides set forth in any one of SEQ ID NOS: <400>1 or <400>3 or <400>5 or <400>7 or any one - 23 -

of SEQ ID NOS:<400>64 to <400>70 or a homologue, derivative or analogue thereof or complementary sequence thereto.

Preferably, the probe is labelled with a reporter molecule capable of giving an identifiable signal (e.g. a radioisotope such as ³²P or ³⁵S or a biotinylated molecule).

An alternative method contemplated in the present invention involves hybridising a nucleic acid primer molecule of at least 10 nucleotides in length derived from an Rp1-D nucleotide sequence or an hrpl nucleotide sequence or Rpg1 nucleotide sequence or a pathogen-resistance gene which is related thereto or a complementary sequence thereto to a nucleic acid "template molecule". Specific nucleic acid molecule copies of the template molecule are amplified enzymatically in a polymerase chain reaction, a technique that is well known to one skilled in the art and described for example by McPherson et al. (1991).

Preferably, the nucleic acid primer molecule or molecule effective in hybridisation is contained in an aqueous mixture of other nucleic acid primer molecules. More preferably, the nucleic acid primer molecule is in a substantially pure form. In a particularly preferred embodiment, the nucleic acid primer molecule is derived from Zea mays, or other monocotyledonous plant such as barley or wheat. In a more preferred embodiment, the nucleic acid primer molecule comprises a nucleotide sequence of at least about 25-30 nucleotides in length derived from, or contained within the nucleotide sequences of the sequences set forth in any one or more of SEQ ID NOS: <400>1 or <400>3 or <400>5 or <400>7 or SEQ ID NOS:<400>64 to <400>70 or a homologue, analogue or derivative thereof.

In a particularly preferred embodiment, the primer and/or probe described according to the preceding embodiments of the present invention comprise a nucleotide sequence set forth in any one of SEQ ID NOS:<400>34 to <400>63.

The nucleic acid template molecule may be in a recombinant form, in a virus particle, - 24 -

bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the related genetic sequence is derived from a monocotyledonous plant such as maize, wheat, barley, triticale, rye, oats, rice or ryegrass and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.

A further aspect of the present invention is directed to a genetic construct which at least comprises an isolated Rp1-D nucleotide sequence or an isolated hrpl nucleotide sequence or an isolated Rpg1 nucleotide sequence or a homologue, analogue or derivative thereof.

Preferably, the Rp1-D nucleotide sequence shares at least about 60% identity to SEQ ID NOS: <400>1 or <400>3 or is a functional derivative, part fragment, homologue, or analogue thereof.

Preferably, the Rpg1 nucleotide sequence shares at least about 60% identity to SEQ ID NOS: <400>5 or <400>7 or is a functional derivative, part fragment, homologue, or analogue thereof.

Preferably, the hrpl nucleotide sequence shares at least about 60% identity to any one of SEQ ID NOS:<400>64 to <400>70 or is a functional derivative, part, fragment, homologue or analogue thereof.

More preferably, the genetic construct comprises the entire open reading frame of an Rp1-D variant gene sequence or the hrpl gene sequence or the Rp1-D variant gene sequence or a fragment thereof which is at least useful as a probe to isolate related sequences.

Alternatively, the subject genetic construct may comprise a region of the open reading frame of an f?p7-D variant or hrpl variant or Rp1-D variant, such as a region which encodes at least about 25 to 30 contiguous amino acids thereof, which is useful for the expression of a recombinant epitope of a pathogen-resistance polypeptide. - 25 -

Accordingly, this embodiment of the present invention clearly extends to genetic constructs for the expression of fusion polypeptides comprising epitopes derived from different pathogen-resistance polypeptides, in particular, fusion polypeptides derived from the Rp1-D and/or Rpg1 variants described herein. Such fusion polypeptides may confer novel resistance phenotypes on transgenic plants in which they are expressed, such as being resistant to different fungal pathogens to the resistance conferred by the base polypeptides from which they are derived.

Those skilled in the art will be aware that recombinant epitopes of a pathogen- resistance polypeptide may also be useful as immunogens for the preparation of antibody molecules, such as polyclonal antibodies, monoclonal antibodies and fragments and immunoglobulin fractions thereof.

Additionally, recombinant Rp1-D and/or Rpg1 epitopes and/or hrpl epitopes and Rp1- D and/or Rpg1 variant nucleotide sequences and/or hrpl variant nucleotide sequences encoding same are useful in screening for the presence and/or expression of pathogen-resistance alleles in plants. The recombinant epitopes and nucleotide sequences may be selected such that they are specific for any particular Rp1-D and/or Rpg1 variant allele and/or hrpl variant allele or alternatively, comprise amino acid sequences common to several different Rp1-D and/or Rpg1 and/or hrpl variant polypeptides.

For example, the present inventors have shown that the Rp1-D allele set forth in SEQ ID NOS: <400>1 and <400>3 shares approximately 58% sequence identity with the corresponding region of the barley Rpg1-2 allele (SEQ ID NO:<400>5) and approximately 60% sequence identity with the corresponding region of the barley Rpg1-13 allele (SEQ ID NO:<400>7), whilst the barley Rpg1-2 and Rpg1-13 alleles are 74% identical overall, wherein higher sequence homology is present in gene regions which encode at least the kinase-1a or p-loop motif core (amino acids 218 to 226 of SEQ ID NO:<400>2 or SEQ ID NO: <400>4), kinase-2a motif core (amino acids 296 to 302 of SEQ ID NO:<400>2 or SEQ ID NO: <400>4), CFL motif (amino acids 447 to - 26 -

453 of SEQ ID NO:<400>2 or SEQ ID NO: <400>4) and WVAEG motif (amino acids 469 to 473 of SEQ ID NO:<400>2 or SEQ ID NO: <400>4) of the nucleotide binding site/leucine-rich repeat (NBS-LRR) proteins encoded by this particular class of disease- resistance genes (i.e. the Rp1-D gene family).

These highly conserved motifs are particularly useful for the generation of immunologically interactive molecules which are capable of binding to an Rp1-D variant polypeptide or Rpg1 variant polypeptide. In an alternative embodiment, Rp1-D and/or Rpg1 nucleotide sequences encoding these highly conserved motifs may be placed operably in connection with a suitable promoter sequence to facilitate the recombinant production of peptides comprising these motifs, optionally to facilitate subsequent antibody production thereto. Those skilled in the art will also be aware that peptides comprising these highly consented motifs may be produced by synthetic means.

The present invention clearly extends to genetic constructs designed to assist expression of an Rp1-D variant polypeptide or Rpg1 variant polypeptide or hrpl variant polypeptide which is capable of conferring, enhancing or facilitating pathogen resistance in a cell or to assist the expression of an immunological epitope or fragment thereof.

Generally, wherein expression of the Rp1-D variant polypeptide or hrpl variant polypeptide or Rpg1 variant polypeptide or epitope or fragment thereof is desired, the genetic construct comprises in addition to the subject nucleic acid molecule, a promoter and optional other regulatory sequences that modulate, regulate or direct expression of the nucleic acid molecule encoding said polypeptide. The promoter may be the f?p7-D gene promoter, the Rpg1 gene promoter or a promoter from another genetic source. Preferably, however, the promoter is capable of expression in a plant cell, either constitutively or in response to infection by a fungal, nematode, viral or bacterial pathogen.

The Rp1-D variant nucleic acid molecule may be genomic DNA or cDNA and may - 27 -

correspond in sequence to the nucleotide sequence set forth in SEQ ID NOS: <400>1 or <400>3 or <400>5 or <400>7 or <400>64 to <400>70 or a homologue, analogue or derivative thereof.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical eukaryotic genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. In the context of the present invention, the term "promoter" also includes the transcriptional regulatory sequences of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or a -10 box transcriptional regulatory sequences.

In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of said sense molecule in a cell. Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of said sense molecule.

Placing a genetic sequence under the regulatory control of a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. A promoter is usually, but not necessarily, positioned upstream or 5' of a nucleic acid molecule which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning - 28 -

of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

Examples of preferred promoters suitable for use in genetic constructs of the present invention include promoters derived from the genes of viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants which are capable of functioning in isolated plant cells and more particularly, monocotyledonous plant cells or whole organisms regenerated therefrom. The promoter may regulate the expression of the pathogen-resistance polypeptide constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others.

Examples of promoters include the CaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, napin seed- specific promoter, P₃₂ promoter, BK5-T imm promoter, lac promoter, tac promoter, phage lambda λ_L or promoters, CMV promoter (U.S. Patent No. 5,168,062), T7 promoter, lacUVδ promoter, SV40 early promoter (U.S. Patent No. 5,118,627), SV40 late promoter (U.S. Patent No. 5,118,627), adenovirus promoter, baculovirus P10 or polyhedrin promoter (U.S. Patent NOS. 5,243,041, 5,242,687, 5,266,317, 4,745,051 and 5,169,784), and the like. In addition to the specific promoters identified herein, cellular promoters for so-called housekeeping genes are useful.

Preferred promoters according to this embodiment are those promoters which are capable of functioning in yeast, mould or plant cells. More preferably, promoters suitable for use according to this embodiment are capable of functioning in cells derived from monocotyledonous plants.

In a more preferred embodiment, the promoter may be derived from a genomic clone - 29 -

encoding a Rp1-D variant polypeptide, preferably derived from the genomic gene set forth in SEQ ID NO: <400>1, more preferably from nucleotides 1 to about 1200 of SEQ ID NO: <400>1 or a homologue, analogue or derivative thereof which is capable of conferring expression on a structural gene in a plant cell.

In an alternative preferred embodiment, the promoter may be derived from a genomic clone encoding an Rpg1 variant polypeptide, preferably derived from the Rpg1-2 allele set forth in SEQ ID NO: <400>5, more preferably from nucleotides 1 to about 3765 of SEQ ID NO: <400>5, or nucleotides from about position 1000 to about 3765 of SEQ ID NO: <400>5, or a homologue, analogue or derivative thereof which is capable of conferring expression on a structural gene in a plant cell.

In an alternative preferred embodiment, the promoter may be derived the Rpg1-13 allele set forth in SEQ ID NO: <400>7, more preferably from nucleotides 1 to about 715 of SEQ ID NO: <400>7 or a homologue, analogue or derivative thereof which is capable of conferring expression on a structural gene in a plant cell.

In a more preferred embodiment, the promoter may be derived from a highly-expressed plant-expressible gene with a view to increasing expression of the pathogen resistance gene to which it is operably connected in the genetic construct.

The genetic construct of the invention may further comprise a terminator sequence and be introduced into a suitable host cell where it is capable of being expressed to produce a recombinant polypeptide gene product.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, - 30 -

fungi, viruses, animals and/or plants.

Examples of terminators particularly suitable for use in the genetic constructs of the present invention include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the ze/t7 gene terminator from Zea mays, the Rubisco small subunit (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, any rΛo-independent E. coli terminator, amongst others.

Those skilled in the art will be aware of additional promoter sequences and terminator sequences which may be suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.

The genetic construct may further comprise a selectable marker gene or genes that are functional in a cell into which said genetic construct is introduced.

As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.

Suitable selectable marker genes contemplated herein include the ampicillin resistance (Amp^r), tetracycline resistance gene (Tfc), bacterial kanamycin resistance gene (Kan"), phosphinothricin resistance gene, neomycin phosphotransferase gene (nptU), hygromycin resistance gene, β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene and luciferase gene, amongst others.

Yet another aspect of the present invention provides for the expression of the subject pathogen-resistance genetic sequence in a suitable host (e.g. a prokaryote or eukaryote) cell, tissue, organ or organism to produce full length or non-full length recombinant pathogen resistance gene products. Preferably, the pathogen resistance - 31 -

gene product has an amino acid sequence that is identical to, or contained within the amino acid sequence set forth in any one of SEQ ID NOS: <400>2 or <400>4 or <400>6 or <400>8, or is at least about 60% identical to at least about 5 contiguous amino acids thereof.

In determining whether or not two amino acid sequences fall within these percentage limits, those skilled in the art will be aware that it is necessary to conduct a side-by-side comparison or multiple alignment of sequences. In such comparisons or alignments, differences will arise in the positioning of non-identical residues, depending upon the algorithm used to perform the alignment. In the present context, reference to a percentage identity or similarity between two or more amino acid sequences shall be taken to refer to the number of identical and similar residues respectively, between said sequences as determined using any standard algorithm known to those skilled in the art. For example, amino acid sequence identities or similarities may be calculated using the GAP programme and/or aligned using the PILEUP programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux etal, 1984). The GAP programme utilizes the algorithm of Needleman and Wunsch (1970) to maximise the number of identical/similar residues and to minimise the number and/or length of sequence gaps in the alignment. Alternatively or in addition, wherein more than two amino acid sequences are being compared, the ClustalW programme of Thompson et al (1994) is used.

The present invention therefore provides a recombinant polypeptide which comprises an amino acid sequence which confers, enhances, or otherwise facilitates resistance to a pathogen in a plant cell, or a functional mutant, homologue, derivative, part, fragment, or analogue of said polypeptide.

In the present context, "homologues" of a polypeptide refer to those polypeptides, enzymes or proteins which have similar properties as a polypeptide of the present invention, for example pathogen-resistance properties in relation to the infection of plants by a fungal pathogen, viral pathogen, nematode pathogen or bacterial pathogen, - 32 -

amongst others, notwithstanding any amino acid substitutions, additions or deletions thereto.

Furthermore, amino acids may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break α-helical structures or β-sheet structures or other conformational structures.

The present invention clearly extends to such amino acid variants, provided that such molecules still function as pathogen-resistance products in conferring, stimulating or otherwise enhancing resistance against a fungal, nematode, viral or bacterial pathogen or alternatively, comprise one or more B cell or T-cell linear or conformational epitopes capable of eliciting the production of antibodies which bind to said pathogen-resistance product or a fragment thereof.

Furthermore, a homologue may be isolated or derived from the same or another plant species as the Rp1-D or Rpg1 variant polypeptides exemplified herein. Preferred sources of homologues of the Zea mays Rp1-D variant set forth in SEQ ID NO: <400>2 or SEQ ID NO: <400>4, or the barley Rpg1 variant polypeptides set forth in SEQ ID NO: <400>6 or SEQ ID NO: <400>8, include any monocotyledonous plant species and more preferably pearl millet, maize, sugar cane, Triticum tauschii, rice, wheat, rye, oats, sorghum, triticale, ryegrass and barley, amongst others.

Furthermore, the amino acids of a homologous polypeptide may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, charge or antigenicity, and so on.

"Analogues" encompass pathogen resistance polypeptides notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein, such as D- stereoisomers and synthetic amino acid analogues. - 33 -

The term "derivative" in relation to a pathogen resistance polypeptide, in particular an Rp1-D or Rpg1 variant polypeptide shall be taken to refer hereinafter to mutants, parts or fragments of a functional molecule. Derivatives include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention. Additionally, derivatives of a pathogen resistance polypeptide may comprise fragments or parts of an amino acid sequence disclosed herein and are within the scope of the invention, as are fusion polypeptides derived from two or more distinct Rp1-D and/or Rpg1 variant polypeptides. Such a fusion polypeptide may provide novel resistance characteristics compared to either Rp1-D and/or Rpg1 variant polypeptides from which it is derived.

Procedures for derivatizing peptides are well-known in the art.

Substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a polypeptide is replaced with another naturally-occurring amino acid of similar character, for example Gly^Ala, Val*-Hle<→Leu, Asp<→Glu, Lys-f→Arg, Asn<→Gln or Phe<-> rp<-> yr.

Substitutions contemplated herein may also be "non-conservative", in which an amino acid residue which is present in a polypeptide is substituted with an amino acid having different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

Amino acid substitutions are typically of single residues, but may be of multiple - 34 -

residues, either clustered or dispersed.

Deletions and insertions may be made to the N-terminus, the C-terminus or be internal deletions or insertions.

The present invention clearly extends to a synthetic peptide fragment of a pathogen resistance gene product, the resistance gene product set forth in any one or more of SEQ ID NOS: <400>2 or <400>4 or <400>6 or <400>8 which may be useful in diagnostic applications or in the generation of antibody molecules.

According to this aspect, the present invention particularly provides a recombinant polypeptide product which is encoded by the Zea mays Rp1-D rust resistance gene or the barley Rpg1 rust resistance gene. This is done, however, with the understanding that the subject invention extends to a range of resistance gene products for rusts and other pathogens of monocotyledonous plants which are encompassed by the terms "Rp1-D variant polypeptide" and/or "Rpg1 variant polypeptide" and/or which satisfy the requirements of a homologue, analogue or derivative of the specific rust-resistance polypeptides exemplified herein.

In fact, the present invention extends to any recombinant polypeptide product of a pathogen resistance gene characterised by said product having at least about 60% similarly to at least about 5 contiguous amino acid residues of SEQ ID NO: <400>2 or SEQ ID NO: <400>4 or SEQ ID NO: <400>6 or SEQ ID NO: <400>8, more preferably at least about 7 contiguous amino acids, even more preferably at least about 9 to 13 contiguous amino acids or at least about 15 to 16 contiguous amino acids or at least about 19 to 20 contiguous amino acids of said amino acid sequences.

Preferably, the recombinant polypeptide of the invention further comprises one or more amino acid sequences selected from: (a) CFLYCSL (SEQ ID NO: <400>9; equivalent to amino acids 447 to 453 of SEQ ID NO: <400>4); and more particularly, LQRCFLYCSLFPKGH (SEQ ID - 35 -

NO: <400>10; equivalent to amino acids 444 to 458 of SEQ ID NO: <400>4) or a homologue, analogue or derivative thereof; and

(b) WXAEG (SEQ ID NO: <400>11 ; equivalent to amino acids 469 to 473 of SEQ ID NO: <400>4); and more particularly, ELVHLW(V/M)AEG (SEQ ID NO: <400>12; equivalent to amino acids 464 to 473 of SEQ ID NO: <400>4) or a homologue, analogue or derivative thereof.

More preferably, the recombinant polypeptide of the present invention further comprises an amino acid sequence motif characterised as a p-Loop, or kinase-1 a motif and having the sequence VGXGGXGKS (SEQ ID NO: <400>13; equivalent to amino acid residues 218 to 226 of SEQ ID NO: <400>4); and more particularly, YSGLAIVGXGGXGKSXLAQ (SEQ ID NO: <400>14; equivalent to amino acid residues 212 to 230 of SEQ ID NO: <400>4) or a homologue, analogue or derivative thereof.

Even more preferably, the pathogen resistance gene product further contains a kinase- 2 motif, having the sequence LLVLDDV (SEQ ID NO: <400>15; equivalent to amino acids 296 to 302 of SEQ ID NO: <400>4); and more particularly FLLVLDDVWFE (SEQ ID NO: <400>16; equivalent to amino acids 294 to 305 of SEQ ID NO: <400>4), or a homologue, analogue or derivative thereof.

In a more particularly preferred embodiment, the recombinant polypeptide of the invention comprises one or more amino acid sequences selected from the list comprising: (a) KYSGLAIVG (SEQ ID NO: <400>17);

(b) YSGLAIVGL (SEQ ID NO: <400>18);

(c) LGGMGKS (SEQ ID NO: <400>19);

(d) GGMGKS (SEQ ID NO: <400>20);

(e) GGMGKST (SEQ ID NO: <400>21); (f) GGMGKSTLAQ (SEQ ID NO: <400>22);

(g) GKSTLAQ (SEQ ID NO: <400>23); - 36 -

(h) STLAQ (SEQ ID NO: <400>24);

(i) TLAQY (SEQ ID NO: <400>25);

0) QKFLLVLDDVWFE (SEQ ID NO: <400>26);

(k) KFLLVLDDVWFEK (SEQ ID NO: <400>27); (I) RLQRCFLYCSLFPKGH (SEQ ID NO: <400>28);

(m) LQRCFLYCSLFPKGHR (SEQ ID NO: <400>29);

(n) ELVHLWVAEG (SEQ ID NO: <400>30);

(o) WVAEG (SEQ ID NO: <400>31);

(p) NELVHLWVAEG (SEQ ID NO: <400>32); and (q) ELVHLWVAEGF (SEQ ID NO: <400>33) or a homologue, analogue or derivative thereof.

The present invention extends to a recombinant gene product that contains the above amino acid sequence motifs in any relative combination or frequency. Preferably the recombinant gene product is capable of conferring, enhancing or facilitating pathogen resistance in a cell.

The recombinant pathogen resistance gene product, pathogen resistance-like gene product, or functional derivative thereof, may be used to produce immunologically interactive molecules, such as antibodies, or functional derivatives thereof, the only requirement being that the recombinant products are immunologically interactive with antibodies to all or part of said gene product.

According to this aspect, there is provided an antibody which is capable of binding to a Rp1-D variant polypeptide wherein said polypeptide comprises substantially the same as the amino acid sequence set forth in SEQ ID NO: <400>2 and/or SEQ ID NO: <400>4 and/or SEQ ID NO: <400>6 and/or SEQ ID NO: <400>8 or is at least about 60% similar to all or at least about 5 contiguous amino acids thereof or an immunologically or functionally equivalent conformational epitope thereof.

Antibodies to a recombinant pathogen resistance gene product are particularly useful - 37 -

in the screening of plants for the presence of said gene product.

Accordingly, antibodies which are capable of binding to the primary amino acid sequence of the pathogen-resistance polypeptide described herein (i.e. a linear epitope) and/or to an epitope thereof which comprises the secondary, tertiary or quaternary structure of said polypeptide (i.e. a conformational epitope) are useful for such applications. Those skilled in the art will be aware that an antibody preparation which is capable of recognising a specific polypeptide may comprise a population of molecules which recognise collectively linear and conformational epitopes.

Antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to a pathogen resistance gene product or may be specifically raised to a recombinant or synthetic pathogen resistance gene product.

The pathogen resistance gene product may first need to be associated with a carrier molecule if it is insufficiently immunogenic without such an association.

Alternatively, fragments of antibodies may be used such as Fab fragments.

Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies and/or the recombinant pathogen resistance gene products of the present invention are particularly useful for the immunological screening of pathogen resistance gene products in various plants, in monitoring expression of pathogen resistance genetic sequences in transgenic plants and as a proprietary tagging system.

In one embodiment, specific antibodies are used to screen for pathogen resistance gene products or pathogen resistance-like gene products in plants. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA. - 38 -

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti- immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of a recombinant pathogen resistance gene product.

Both polyclonal and monoclonal antibodies are obtainable by immunisation with a recombinant pathogen resistance gene product and either type is utilisable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of recombinant pathogen resistance gene product, or antigenic or immunointeractive parts thereof, collecting serum from the animal and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilisable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitised against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art (see, for example, Douillard and Hoffman, 1981 ; Kohler and Milstein, 1975).

The presence of a pathogen resistance gene product or pathogen resistance-like gene product in a plant or more commonly a plant extract may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent NOS. 4,016,043, 4, 424,279 and 4,018,653. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the - 39 -

traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilised on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time and under conditions sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule.

In this case, the first antibody is raised to a recombinant pathogen resistance gene product and the antigen is a pathogen resistance gene product in a plant.

The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain pathogen resistance gene product and include crude or purified plant extract such as extracts of leaves, roots and stems.

In the typical forward sandwich assay, a first antibody raised against a recombinant pathogen resistance gene product is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used - 40 -

polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking, covalent binding or physically adsorption, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25°C) to allow binding of any antigen present in the sample to the antibody. Following the incubation period, the reaction locus is washed and dried and incubated with a second antibody specific for a portion of the first antibody. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilising the target molecules in the biological sample and then exposing the immobilised target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detected by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily - 41 -

recognised, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. The term "reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescem and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in enzyme immunoassays (EIA), the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. - 42 -

It will be readily apparent to the skilled technician how to vary the above assays and all such variations are encompassed by the present invention.

In an alternative embodiment, the hybridisation and PCR techniques described supra may be utilised with any necessary modifications to determine the presence of a pathogen-resistance gene in a plant or to quantitate the level of expression of said gene at the RNA level.

Those skilled in the art will be aware that the genetic construct described supra may be used to "transfect" a cell, in which case it is introduced into said cell without integration into the cell's genome. Alternatively, a genetic construct may be used to "transform" a cell, in which case it is stably integrated into the genome of said cell.

Accordingly, the isolated nucleotide sequence of the present invention, or homologue, analogue or derivative thereof, may be introduced into a cell using any known method for the transfection or transformation of said cell, thereby conferring enhanced pathogen-resistance properties thereon. Wherein a cell is transformed by the genetic construct of the invention, a whole organism may be regenerated from a single transformed cell, using any method known to those skilled in the art.

Accordingly, a further aspect of the invention extends to a plant such as a crop plant carrying a non-endogenous Rp1-D variant or Rpg1 variant which encodes a polypeptide which confers, enhances, or otherwise facilitates pathogen resistance in said plant. Preferably, the plant is a monocot plant. More preferably the transgenic plant is one or more of the following: wheat, maize, barley, rye, oats, pearl millet, rice, sorghum, sugar cane, Triticum tauschii or ryegrass, amongst others. Other species are not excluded.

In fact, the non-endogenous Rp1-D variant genetic sequence, Rpg1 variant genetic sequence or transgene may originate from any plant species. Preferably, said genetic sequence is identical to any one or more of the nucleotide sequences set forth in SEQ - 43 -

ID NOS: <400>1 or <400>3 or <400>5 or <400>7, or <400>64 to <400>70 or a functional derivative, fragment, part, complement, homologue, or analogue thereof.

Furthermore, wherein said genetic sequence or transgene is a cDNA molecule such as set forth in SEQ ID NO: <400>3 or other nucleic acid molecule which lacks a functional promoter, it may be placed operably under control of promoter sequence. The expression of the transgene may be constitutive or inducible by an external stimulus such as physiological stress, or by addition of a chemical compound, or the expression may be developmentally-regulated, or expressed in a tissue- or cell-specific pattern. Furthermore the transgene may be inserted into or fused to a particular endogenous genetic sequence. Methods for placing a structural gene operably under the control of a promoter sequence are well-known to those skilled in the art.

A genetic construct designed to express a recombinant Rp1-D variant polypeptide or variant Rpg1 polypeptide may be introduced into plant tissue, thereby producing a "transgenic plant", by various techniques known to those skilled in the art. The technique used for a given plant species or specific type of plant tissue depends on the known successful techniques. Means for introducing recombinant DNA into plant tissue include, but are not limited to, direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990) microparticle bombardment electroporation (Fromm et al., 1985), microinjection of DNA (Crossway et al., 1986), microparticle bombardment of tissue explants or cells (Christou etal, 1988; Sanford, 1988) or T-DNA-mediated transfer from Agrobacterium to the plant tissue. Methods for the Agro- acter/^'um-mediated transformation of plants will be well-known to those skilled in the art. In particular, methods for the Agro- acter/utrj-mediated transformation of rice {Oryza sativa) tissue have been disclosed by Heie et al.. Representative T-DNA vector systems are described in the following references: An et a/.(1985); Herrera-Estrella etal. (1983a,b); Herrera-Estrella et al. (1985).

For microparticle bombardment of cells, a microparticle is propelled into a plant cell, - 44 -

in particular a plant cell not amenable to Agrobacten^'um mediated transformation, to produce a transformed cell. Wherein the cell is a plant cell, a whole plant may be regenerated from the transformed plant cell. Alternatively, other non-animal cells derived from multicellular species may be regenerated into whole organisms by means known to those skilled in the art. Any suitable ballistic cell transformation methodology and apparatus can be used in practising the present invention. Exemplary apparatus and procedures are disclosed by Stomp etal. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No.4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.

Examples of microparticles suitable for use in such systems include 1 to 5 μm gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

Plant species may be transformed with the genetic construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art.

Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector of the present invention. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

The term "organogenesis", as used herein, means a process by which shoots and roots are developed sequentially from meristematic centres. - 45 -

The term "embryogenesis", as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.

Plants of the present invention may take a variety of forms. The plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the expression cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species). The transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.

The genetic construct may further incorporate a dominant selectable marker, such as npt\\, hygromycin-resistance gene, a phosphinothricin-resistance gene or ampicillin- resistance gene, amongst others, associated with the transforming DNA to assist in cell selection and breeding.

Once introduced into the plant tissue, the expression of the introduced gene may be assayed in a transient expression system, or it may be determined after selection for stable integration within the plant genome. Techniques are known for the in vitro culture of plant tissue, and in a number of cases, for regeneration into whole plants. Procedures for transferring the introduced genetic construct from the originally transformed plant into commercially useful cultivars are known to those skilled in the art.

In the context of the present invention, it is preferred that the transgenic plants thus generated exhibit enhanced pathogen-resistance properties compared to otherwise isogenic non-transformed plants, in particular against fungal pathogens and viral pathogens. For example, Rp1-D and/or Rpg7 and/or hrpl variant genes which confer - 46 -

or enhance rust-resistance can be introduced into cereals to produce novel lines with improved resistance to rusts, as an alternative or adjunct to conventional plant breeding approaches. In particular, Rp1-D and/or other Rp alleles may be used to confer resistance against rusts such as Puccinia ssp. in maize, sweet corn, wheat and sorghum, amongst others. Similarly, the barley Rpg7 variant alleles exemplified herein may be used to confer resistance against barley stem rust, amongst others. Whilst rice does not have a rust pathogen, it does contain Rp1-D variant genes which may be useful in conferring resistance against rice pathogens such as the rice blast Pyricularia oryzae or the rice blight Xanthomonas oryzae, amongst others. Similarly, the Rp1-D variant of wheat may be used to confer resistance against wheat streak mosaic virus, amongst others in cereals.

The present invention extends to the progeny and clonal derivatives of said transgenic plant.

The present invention is further described in the following Examples. The embodiments exemplified hereinafter are in no way to be taken as limiting the subject invention.

EXAMPLE 1 Strategy for isolating Rp1-D variant genes

The Rp1-D rust resistance gene was isolated from maize by transposon tagging using two independent maize transposon systems (Mu and Ac7Ds). The two methods resulted in the isolation of the same identical DNA sequence: the Rp1-D gene, which belongs to the NBS-LRR class of plant resistance gene.

The Rp1-D rust resistance gene was isolated from maize after tagging with the maize transposon Mu. A DNA fragment, identified with a Mu probe, is absent in the rust resistant parent and present in the susceptible transposon-tagged mutant. This new - 47 -

fragment co-segregates with the mutant Rp1-D allele. DNA flanking the Mu insertion in the novel fragment was cloned and sequenced. The putative translation product of the flanking DNA sequence was shown to encode a leucine-rich repeat nucleotide binding site polypeptide (SEQ ID NO:<400>2 or SEQ ID NO: <400>4).

In a further approach, the Rp1-D rust resistance gene was isolated from maize after tagging with the maize transposon Ds. A mutant allele was identified and cloned using a DNA probe encoding a resistance gene analogue sequence derived by PCR using pools of degenerate primers designed to encode conserved amino acid sequence motifs present in NBS-LRR resistance genes (Table 2). The Ds element is absent in the wild-type allele, appears in the mutant allele and is absent in rust resistant revertants of the Ds allele.

The Ds containing allele has the identical sequence of the gene tagged by the Mu transposon. Both strategies used to isolate the Zea mays Rp1-D allele set forth in SEQ ID NO: <400>1 thus relied upon the transposon mutagenesis of Zea mays and required detailed genetic data to facilitate linkage analysis demonstrating co- segregation of the resistance phenotype or susceptible phenotype with the wild-type allele or mutant allele, respectively. In this regard, absent any linkage data mere amplification approaches alone would have presented considerable difficulties in assigning a function to the amplified NBS-LRR encoding nucleotide sequences obtained, particularly in consideration of the diversity of such sequences present in plant genomes. Notwithstanding that this is the case, those skilled in the art will recognise that once the function of the nucleotide sequence of the Zea mays Rp1-D allele set forth in SEQ ID NOS: <400>1 and <400>3 was established by the present inventors, said sequence provided a means for isolating and/or detecting functionally- equivalent sequences in Zea mays or other plants.

EXAMPLE 2 Isolation of Rp1~D sequences using the Mutator transposable element system

Families which were heterozygous for two different Rp1 alleles (Rp1-D/Rp1-A and - 48 -

Rp1-D/Rp1-B) with multiple, active Mutator transposable elements were constructed and out crossed to Rp1-J homozygotes. The resulting families were screened with the common rust (P.sorghi) biotype IN2, which is virulent on lines carrying Rp1-J, but avirulent on lines carrying Rp1-D, Rp1-A and Rp1-B. After testing 150,000 maize seedlings with biotype IN2, 55 susceptible individuals were identified. DNA from the susceptible individuals were analysed with the RFLP probes umc285 and bnl3.04 which detect RFLP loci which flank the locus. This permitted the distinction between susceptible individuals which arose from cross over events from those which did not, the latter being candidates for transposon insertion mutants. Four of the susceptible individuals had flanking RFLP marker alleles of the Rp7-D parent, indicating they were derived from mutations of the Rp1-D allele and were not derived from crossing-over. Each of the four mutant individuals was crossed two or three times to maize lines which carried detectable rp1 alleles but no Mutator elements to reduce the number of Mutator elements in the line. DNAs of families derived from each of the four mutants, segregating for the mutant allele, were then analysed by gel blot analysis with several Mutator DNA probes to determine if any of the lines carried Mutator elements which mapped to the rp1 locus. No mutator elements which mapped to the rp1 locus were identified in three of the four families.

In the family segregating for the fourth mutation, a Mutator probe hybridized to a Hind U fragment of approximately 5 Kb which cosegregated with the mutant allele in 90 progeny. After self-fertilizing an individual carrying the mutant allele, an individual which was homozygous for the mutant allele was identified, DNA was purified and a library of 4-6 kb Hind\\\ fragments was made in a Lambda cloning vector. The 5 Kb Hind\\\ fragment which mapped to rp1 was selected using a Mutator probe and the 5 Kb Hind\\\ fragment was sequenced. The clone was found to carry a Mu2 element inserted into approximately 3 Kb of DNA with sequence homology to known resistance genes.

When used as a probe in gel-blot analysis, the sequences flanking the Mu2 element detect a gene family which maps to the rp1 locus. 49 -

TABLE 2 OLIGONUCLEOTIDE PRIMERS USED TO AMPLIFY Rp1 ALLELES

Primer Primer sequence (5' to 3') SEQ ID NO: p-loopAA* AAG AAT TCG GNG TNG GNA AAA CAA C <400>34 p-loopAT* AAG AAT TCG GNG TNG GNA AAA CTA C <400>35 p-loopAC^* AAG AAT TCG GNG TNG GNA AAA CCA C <400>36 p-loopAG* AAG AAT TCG GNG TNG GNA AAA CGA C <400>37 p-loopGA* AAG AAT TCG GNG TNG GNA AGA CAA C <400>38 p-loopGT* AAG AAT TCG GNG TNG GNA AGA CTA C <400>39 p-loopGC* AAG AAT TCG GNG TNG GNA AGA CCA C <400>40 p-loopGG* AAG AAT TCG GNG TNG GNA AGA CGA C <400>41 kinase-2D CTA CTG NTN CTN GAC GAC GT <400>42 kinase-2E CTA CTG NTN CTN GAC GAT GT <400>43 kinase-2F CTA CTG NTN CTN GAT GAC GT <400>44 kinase-2G CTA CTG NTN CTN GAT GAT GT <400>45

GLPL1* AAC TCG AGA GNG CNA GNG GNA GGC C <400>46 GLPL2^* AAC TCG AGA GNG CNA GNG GNA GAC C <400>47 GLPL3^* AAC TCG AGA GNG CNA GNG GNA GTC C <400>48 GLPL4* AAC TCG AGA GNG CNA GNG GNA GCC C <400>49 GLPL5^* AAC TCG AGA ANG CCA ANG GCA ATC C <400>50 GLPL6* AAC TCG AGA ANG CCA ANG GCA AAC C <400>51

CFA1 CA(A/G) (T/A)AI GC(G/A) AA(G/A) CA(C/T) TGT TT <400>52 CFA2 CA(A/G) (T/A)AI GC(G/A) AA(G/A) CA(C/T) TGC TT <400>53 CFA3 ATA GA(A/G) CA(A/G) (T/A)AI GC(G/A) AAA CA <400>54 CFA4 ATA GA(A/G) CA(A/G) (T/A)AI GC(G/A) AAG CA <400>55

WMA1 A(T/C)(A/G) AAN CCN TNA GCC ATC CA <400>56 WMA2 A(T/C)(A/G) AAN CCN TNT GCC ATC CA <400>57 WMA3 A(T/C)(A/G) AAN CCN TNC GCC ATC CA <400>58 W A4 A(T/C)(A/G) AAN CCN TNG GCC ATC CA <400>59

50 -

MHD1 CGA CAG TCN ATC ATG CAT <400>60 MHD2 CGA CAG TCN ATC GTG CAT <400>61 MHD3 CGA CAG TCN GTC ATG CAT <400>62 MHD4 CGA CAG TCN GTC GTG CAT <400>63 ^* Primers are the same as those described by R. Michelmore (University of California, Davis) except for the 5'ends. Primer names accompanied by an asterisk to indicate these differences.

EXAMPLE 3 Protocol for the isolation of Rp1-D sequences using the Ds transposable element system

Plants that were homozygous for the Rp1-D13 allele and heterozygous for the Pw Ac containing gene were crossed by plants containing the Rp5 rust resistance gene which is linked and about 1-2 cm distal on the short arm of chromosome 10 (Figure 1). A number of susceptible mutants were recovered from 10,000 progeny screened. These were selfed and used to isolate homozygous mutants of the Rp1-D13 allele. Several of these were tested for reversion to resistance in the presence of Ac activity. The susceptible mutant rp1-D13-2 reverted to resistance in 2/2800 test cross progeny providing genetic evidence for tagging with a Ds transposon. The PIC20 DNA clone, containing a resistance gene analogue sequence, reveals a small gene family that in Southern analysis co-segregates with the rp1 locus (Figures 2A and 2B). When used for analysis of the tpM 3-2 mutant and resistant revertants, the PCI20 probe demonstrated the presence of an approximately 400 bp insertion in rp1-D-13-2 which was absent in the resistant revertants (Figure 2A, compare lanes 22 and 23). Cloning and sequencing of the band with the altered size confirmed the presence of the Ds insertion and the flanking DNA sequence was shown to be identical to the sequences isolated independently by the Mu transposon tagging experiment.

EXAMPLE 4 Rp1-D DNA Probes can detect and clone resistance genes in other cereals A DNA probe isolated from the nucleotide binding site region of the rp1 gene detected RFLPs between barley varieties Steptoe and Morex (Figure 3). Morex contains the - 51 -

Rpg1 rust resistance gene and is resistant to barley stem rust, while Steptoe lacks this gene and is susceptible. A mapping family derived from doubled haploids of a Steptoe X Morex F1 plant (Ref) which has been scored for Rpg1 resistance was used to map the rp1 RFLPs. One RFLP co-segregated for Rpg1 in 150 progeny (Figure 3). Two other unlinked loci were also detected with the Rp1 probe which may correspond to other cereal disease resistance loci. Two alleles of the gene family at the Rpg1 locus were cloned and sequenced, designated Rpg1-2 (SEQ ID NO:<400>5) and Rpg1-13 (SEQ ID NO:<400>7). ClustalW analyses indicate significant homologies between the Rp1-D, Rpg1-2 and Rpg1-13 alleles, at both the nucleotide level and the amino acid level (Figures 4A and 5). The Rpg1-2 allele is 58% identical to Rp1-D at the protein level, whilst the Rpg1-13 allele is 60% identical to Rp1-D (Figure 4A; Figure 5). The Rpg1-2 and Rpg1-13 proteins are 74% identical. Accordingly, the term "at least about 60% identical" shall be understood to include 57% or 58% or 59% or 60% identity.

EXAMPLE 5

Identification of Rp1-D variants in other plant species

Genomic DNA was isolated from seedlings of sorghum, Panicum ssp., maize, sugar cane, barley, hexaploid wheat, oats, Triticum tauschii and rice, digested with either Λ/col or Sg/ll and subjected to Southern hybridisation using a maize Rp1-D nucleotide sequence as a probe. As shown in Figure 6, hybridising bands were present in all species tested, suggesting that Rp1-D variant alleles are present in a wide range of different monocotyledonous plants.

EXAMPLE 6 Coding regions of Rp1-D homologous genes

Rp1-D homologous genes were cloned from Zea mays and the coding regions determined. The nucleotide sequences of these coding regions are shown in SEQ ID NOS:<400> 64 to <400> 70. The homologous genes are referred to herein as hrpl- d1 to hrp1-d6 (SEQ ID NOS:<400>64 to <400>69, respectively and hrp1-cin4 (SEQ ID NO:<400>70). - 52 -

REFERENCES

1. An et al. (1985) EMBO J 4:277-284.

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3. Ausubel, et al (1992), Current Protocols in Molecular Biology, Greene/Wiley, New York.

4. Christou, P., et al. Plant Physiol 87: 671-674, 1988.

5. Crossway et al., Mol. Gen. Genet. 202:179-185, 1986.

6. Devereux, J., et al. (1984). Nucl. Acids Res. 12:387-395.

7. Dixon, R., and Lamb, C (1990) Ann. Rev. Plant Physiol. Plant Mol. Biol. 41:339- 367.

8. Douillard and Hoffman (1981) In: Compendium of Immunology Vol II (ed. Schwarz).

9. Fromm et al. Proc. Natl. Acad. Sci. (USA) 82:5824-5828, 1985.

10. Herrera-Estella et al. , Nature 303: 209-213, 1983a.

11. Herrera-Estella et al.,EMBO J. 2: 987-995, 1983b.

12. Herrera-Estella et al. In: Plant Genetic Engineering, Cambridge University Press, N.Y., pp 63-93, 1985.

13. Kohler and Milstein (1975) Nature 256: 495-499.

14. Krens, F.A., et al., Nature 296: 72-74, 1982.

15. Marineau, C, Matton, D.P. and Brisson, N. (1987) Plant Mol. Biol. 9: 335-342.

16. McPherson, M.J., Quirke, P. and Taylor, G.R. (1991) PCR A Practical Approach. IRL Press, Oxford University Press, Oxford, United Kingdom.

17. Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453.

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19. Sambrook et al (1989), Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory Press

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21. Thompsons al., (1994) Nucl. Acids Res. 22:4673-4680.

Claims

- 53 -CLAIMS:

1. An isolated nucleic acid molecule comprising an Rp1-D nucleotide sequence or Rpg1 nucleotide sequence OR hrpl nucleotide sequence or a variant thereof which encodes a protein or derivative thereof, which confers, enhances, or otherwise facilitates resistance to a pathogen in a plant.

2. The isolated nucleic acid molecule according to claim 1 , wherein the Rp1-D nucleotide sequence or hrpl nucleotide sequence or variant thereof comprises a nucleotide sequence that is at least 60% identical to any one of SEQ ID NO:<400>1 or SEQ ID NO:<400>3 or SEQ ID NOS:<400>64 to <400>70 or a complementary nucleotide sequence thereto.

3. The isolated nucleic acid molecule according to claim 2, wherein the variant is derived from a monocotyledonous plant.

4. The isolated nucleic acid molecule according to claim 3, wherein the monocotyledonous plant is selected from the list comprising Zea mays, pearl millet, sugar cane, wheat, barley, rye, rice, oats, triticale, sorghum and ryegrass.

5. The isolated nucleic acid molecule according to claim 4, wherein the monocotyledonous plant is Zea mays or barley.

6. The isolated nucleic acid molecule according to claim 1 , wherein the Rp1-D nucleotide sequence or variant thereof comprises the nucleotide sequence set forth in any one of SEQ ID NO:<400>1 or SEQ ID NO:<400>3 or SEQ ID NOS:<400>64 to <400>7 or a derivative of said nucleotide sequence having comprising at least about 30 contiguous nucleotides of any one of SEQ ID NOS: <400>1 and/or <400>3 and/or SEQ ID NOS:<400>64 to <400>70.

7. The isolated nucleic acid molecule according to claim 1 , wherein the Rpg1 nucleotide sequence or variant thereof comprises a nucleotide sequence that is at - 54 -

least 60% identical to SEQ ID NO:<400>5 or a complementary nucleotide sequence thereto.

8. The isolated nucleic acid molecule according to claim 7, wherein the Rpg1 variant is derived from a monocotyledonous plant.

9. The isolated nucleic acid molecule according to claim 8, wherein the monocotyledonous plant is selected from the list comprising Zea mays, pearl millet, sugar cane, wheat, barley, rye, rice, oats, triticale, sorghum and ryegrass.

10. The isolated nucleic acid molecule according to claim 9, wherein the monocotyledonous plant is Zea mays or barley.

11. The isolated nucleic acid molecule according to claim 1 , wherein the Rpg nucleotide sequence or variant thereof comprises the nucleotide sequence set forth in SEQ ID NO:<400>5 or a derivative of said nucleotide sequence having comprising at least about 30 contiguous nucleotides of SEQ ID NO: <400>5.

12. The isolated nucleic acid molecule according to claim 1 , wherein the Rpg nucleotide sequence or variant thereof comprises a nucleotide sequence that is at least 60% identical to SEQ ID NO:<400>7 or a complementary nucleotide sequence thereto.

13. The isolated nucleic acid molecule according to claim 12, wherein the Rpg1 variant is derived from a monocotyledonous plant.

14. The isolated nucleic acid molecule according to claim 13, wherein the monocotyledonous plant is selected from the list comprising Zea mays, pearl millet, sugar cane, wheat, barley, rye, rice, oats, triticale, sorghum and ryegrass.

15. The isolated nucleic acid molecule according to claim 14, wherein the - 55 -

monocotyledonous plant is Zea mays or barley.

16. The isolated nucleic acid molecule according to claim 15, wherein the Rpg1 nucleotide sequence or variant thereof comprises the nucleotide sequence set forth in SEQ ID NO:<400>7 or a derivative of said nucleotide sequence having comprising at least about 30 contiguous nucleotides of SEQ ID NO: <400>7.

17. An isolated nucleic acid molecule which encodes a protein or derivative thereof, which confers, enhances, or otherwise facilitates resistance to a pathogen in a plant, wherein said nucleic acid molecule comprises a nucleotide sequence which hybridises under at least low stringency conditions to at least about 25-30 contiguous nucleotides of SEQ ID NOS: <400>1 and/or <400>3 and/or <400>5 and/or <400>7 and/or any one of SEQ ID NOS:<400>64 to <400>70 or a complementary sequence thereto.

18. The isolated nucleic acid molecule according to claim 17, derived from a monocotyledonous plant.

19. The isolated nucleic acid molecule according to claim 18, wherein the monocotyledonous plant is selected from the list comprising Zea mays, pearl millet, sugar cane, wheat, barley, rye, rice, oats, triticale, sorghum and ryegrass.

20. The isolated nucleic acid molecule according to claim 19, wherein the monocotyledonous plant is Zea mays or barley.

21. The isolated nucleic acid molecule according to claim 17 comprising the nucleotide sequence set forth in SEQ ID NOS: <400>1 and/or <400>3 and/or any one of SEQ ID NOS:<400>64 to <400>70 or a complementary sequence thereto.

22. The isolated nucleic acid molecule according to claim 17 comprising the nucleotide sequence set forth in SEQ ID NO: <400>5 or a complementary sequence thereto. - 56 -

23. The isolated nucleic acid molecule according to claim 17 comprising the nucleotide sequence set forth in SEQ ID NO: <400>7 or a complementary sequence thereto.

24. An isolated nucleic acid molecule which encodes a pathogen-resistance protein or derivative thereof, which confers, enhances, or otherwise facilitates resistance to a pathogen in a plant, wherein said pathogen-resistance protein or derivative comprises an amino acid sequence set forth in any one of SEQ ID NOS: <400>2, <400>4, <400>6 or <400>8 or a homologue, analogue or derivative of said amino acid sequences that is at least about 60% identical thereto.

25. The isolated nucleic acid molecule according to claim 24, derived from a monocotyledonous plant.

26. The isolated nucleic acid molecule according to claim 25, wherein the monocotyledonous plant is selected from the list comprising Zea mays, pearl millet, sugar cane, wheat, barley, rye, rice, oats, triticale, sorghum and ryegrass.

27. The isolated nucleic acid molecule according to claim 26, wherein the monocotyledonous plant is Zea mays or barley.

28. The isolated nucleic acid molecule according to claim 24, wherein the pathogen- resistance protein or derivative comprises the amino acid sequence set forth in SEQ ID NOS: <400>2 or <400>4 or at least 10 contiguous amino acids thereof.

29. The isolated nucleic acid molecule according to claim 24, wherein the pathogen- resistance protein or derivative comprises the amino acid sequence set forth in SEQ ID NO: <400>6 or at least 10 contiguous amino acids thereof.

30. The isolated nucleic acid molecule according to claim 24, wherein the pathogen- resistance protein or derivative comprises the amino acid sequence set forth in SEQ - 57 -

ID NO: <400>8 or at least 10 contiguous amino acids thereof.

31. The isolated nucleic acid molecule according to claim 24, wherein the pathogen- resistance protein or derivative further includes one or more of the amino acid sequences set forth in any one of SEQ ID NOS: <400>17 to <400>33.

32. A method of identifying a pathogen resistance genetic sequence or pathogen resistance-like genetic sequence in a plant, said method comprising contacting genomic DNA, mRNA, cDNA or a fragment or a source thereof, with a hybridisation- effective amount of a probe comprising at least about 25-30 contiguous nucleotides of any one of SEQ ID NOS: <400>1 or <400>3 or <400>5 or <400>7 or any one of SEQ ID NOS:<400>64 to <400>70 or a complementary nucleotide sequence thereto for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation.

33. The method according to clam 32 wherein the probe is labelled with a reporter molecule.

34. The method according to clam 32 wherein the plant is a broadacre crop plant and/or a monocotyledonous plant.

35. The method according to claim 34, wherein the monocotyledonous plant is maize, barley, rye, oat, wheat, sorghum, triticale, ryegrass or rice or a wild variety and/or hybrid or derivative and/or ancestral progenitor of same.

36. A method of identifying a pathogen resistance genetic sequence or pathogen resistance-like genetic sequence in a plant, said method comprising hybridising one or more nucleic acid primer molecules of at least 10 nucleotides in length of any one of SEQ ID NOS: <400>1 or <400>3 or <400>5 or <400>7 or any one of SEQ ID NOS:<400>64 to <400>70 or a complementary nucleotide sequence thereto with genomic DNA, mRNA, cDNA or a fragment or a source thereof, and amplifying nucleic - 58 -

acid therefrom in a polymerase chain reaction.

37. The method according to claim 36 wherein the primer comprises a nucleotide sequence set forth in any one of SEQ ID NOS:<400>34 to <400>63.

38. The method according to clam 36 wherein the plant is a broadacre crop plant and/or a monocotyledonous plant.

39. The method according to claim 38, wherein the monocotyledonous plant is maize, barley, rye, oat, wheat, sorghum, triticale, ryegrass or rice or a wild variety and/or hybrid or derivative and/or ancestral progenitor of same.

40. A genetic construct that comprises the isolated nucleic acid molecule according to claim 1 or the protein-encoding region thereof in operable connection with a promoter sequence.

41. The genetic construct according to claim 40, wherein the promoter sequence is operable in a plant cell, tissue, organ or whole plant.

42. The genetic construct according to claim 41 , wherein the promoter sequence comprises nucleotides 1 to about 1200 of SEQ ID NO: <400>1 or nucleotides 1 to about 3765 of SEQ ID NO: <400>5, or nucleotides 1 to about 715 of SEQ ID NO: <400>7 or a homologue, analogue or derivative thereof which is capable of conferring expression on a structural gene in a plant cell.

43. The genetic construct according to claim 41 , wherein the promoter sequence comprises the CaMV 35S promoter, NOS promoter, OCS promoter, A. thaliana SSU promoter or napin promoter sequence.

44. An isolated promoter sequence that is capable of conferring expression on a structural gene in a plant cell comprising nucleotides 1 to about 1200 of SEQ ID NO: <400>1. - 59 -

45. An isolated promoter sequence that is capable of conferring expression on a structural gene in a plant cell comprising nucleotides 1 to about 3765 of SEQ ID NO: <400>5.

46. An isolated promoter sequence that is capable of conferring expression on a structural gene in a plant cell comprising nucleotides 1 to about 715 of SEQ ID NO: <400>7.

47. A pathogen resistance protein substantially free of conspecific proteins and having at least about 60% amino acid sequence identity to any one or more of SEQ ID NOS:<400>2, <400>4, <400>6 or <400>8, wherein said pathogen resistance protein confers, enhances, or otherwise facilitates resistance to a pathogen in a plant.

48. The pathogen resistance protein according to claim 47 comprising the amino acid sequence set forth in SEQ ID NOS: <400>2 or <400>4 or at least 10 contiguous amino acids thereof.

49. The pathogen resistance protein according to claim 47 comprising the amino acid sequence set forth in SEQ ID NO: <400>6 or at least 10 contiguous amino acids thereof.

50. The pathogen resistance protein according to claim 47 comprising the amino acid sequence set forth in SEQ ID NO: <400>8 or at least 10 contiguous amino acids thereof.

51. The pathogen resistance protein according to claim 47 further including one or more of the amino acid sequences set forth in any one of SEQ ID NOS: <400>17 to <400>33.

52. An antibody molecule that binds to the pathogen resistance protein according to claim 47 or an epitope thereof. - 60 -

53. A method of producing a plant having improved resistance to a plant pathogen comprising expressing a pathogen resistance protein which comprises an amino acid sequence having at least about 60% amino acid sequence identity to any one or more of SEQ ID NOS:<400>2, <400>4, <400>6 or <400>8 in said plant or a cell, tissue or organ thereof.

54. The method according to claim 53, further comprising introducing a nucleic acid molecule encoding the pathogen resistance protein into a plant cell, tissue, organ or whole plant in a format suitable for expression to occur.

55. The method according to claim 53, further comprising introducing a nucleic acid molecule encoding the pathogen resistance protein into a plant cell, tissue or organ in a format suitable for expression to occur and regenerating a whole plant therefrom.

57. The method according to claims 54 or 55, wherein the nucleic acid molecule is in operable connection with a promoter sequence capable of being expressed in a plant cell, tissue, organ or whole plant.

58. The method according to claim 53, wherein the pathogen resistance protein comprises an amino acid sequence set forth in SEQ ID NOS:<400>2 or <400>4.

59. The method according to claim 53, wherein the pathogen resistance protein comprises an amino acid sequence set forth in SEQ ID NO:<400>6.

60. The method according to claim 53, wherein the pathogen resistance protein comprises an amino acid sequence set forth in SEQ ID NO:<400>8.

61. The method according to claim 53 wherein the plant pathogen is a fungus.

62. The method according to claim 53 wherein the plant pathogen is a rust. - 61 -

63. The method according to claim 62 wherein the rust is Puccinia sp.

64. The method according to claim 62 wherein the rust is barley stem rust.

65. The method according to claim 62 wherein the rust is Pyricularia oryzae.

66. The method according to claim 62 wherein the rust is Xanthomonas oryzae.

67. The method according to claim 53 wherein the plant pathogen is a virus.

68. The method according to claim 67 wherein the virus is wheat streak mosaic virus.

69. The method according to claim 53 wherein the plant pathogen is a bacterium.

70. The method according to claim 53 wherein the plant pathogen is a nematode.

71. A plant produced by the method comprising:

(i) introducing a nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID NOS: <400>1 or <400>3 or <400>5 or <400>7 or any one of SEQ ID NOS:<400>64 to <400>70 or a protein-encoding fragment thereof into a plant cell, tissue or organ in a format suitable for expression to occur; and (ii) regenerating a whole plant therefrom.

72. The plant according to claim 71 capable of expressing the pathogen resistance protein or a biologically active fragment thereof encoded by the introduced nucleic acid molecule.

73. The plant according to claim 72 having improved resistance to a plant virus, fungus, bacteria, nematode or rust pathogen. - 62 -

74. The plant according to claim 73 wherein the rust is Puccinia sp.

75. The plant according to claim 73 wherein the rust is barley stem rust.

-63- AMENDED CLAIMS

[received by the International Bureau on 26 May 1999 (26.05.99); original claims 57 - 75 replaced by new claims 56 -74 (3 pages)]

56. The method according to claims 54 or 55, wherein the nucleic acid molecule is in operable connection with a promoter sequence capable of being expressed in a plant cell, tissue, organ or whole plant.

57. The method according to claim 53, wherein the pathogen resistance protein comprises an amino acid sequence set forth in SEQ ID NOS:<400>2 or <400>4.

58. The method according to claim 53, wherein the pathogen resistance protein comprises an amino acid sequence set forth in SEQ ID NO:<400>6.

59. The method according to claim 53, wherein the pathogen resistance protein comprises an amino acid sequence set forth in SEQ ID NO:<400>8.

60. The method according to claim 53 wherein the plant pathogen is a fungus.

61. The method according to claim 53 wherein the plant pathogen is a rust. -64-

62. The method according to claim 61 wherein the rust is Puccinia sp.

63. The method according to claim 61 wherein the rust is barley stem rust.

64. The method according to claim 61 wherein the rust is Pyricularia oryzae.

65. The method according to claim 61 wherein the rust is Xanthomonas oryzae.

66. The method according to claim 53 wherein the plant pathogen is a virus.

67. The method according to claim 66 wherein the virus is wheat streak mosaic virus.

68. The method according to claim 53 wherein the plant pathogen is a bacterium.

69. The method according to claim 53 wherein the plant pathogen is a nematode.

70. A plant produced by the method comprising:

71. The plant according to claim 70 capable of expressing the pathogen resistance protein or a biologically active fragment thereof encoded by the introduced nucleic acid molecule.

72. The plant according to claim 71 having improved resistance to a plant virus, fungus, bacteria, nematode or rust pathogen. -65-

73. The plant according to claim 72 wherein the rust is Puccinia sp.

74. The plant according to claim 72 wherein the rust is barley stem rust.