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AssigneeNone recorded
InventorsPaula Maria Josef Schulze-Lefert, Joachim Kurth, Zhou Fasong, Candace Elliott, Roger Philip Wise, Dennis Allen Halterman, Fusheng Wei
Estimating the term…
Disclosed are isolated nucleic acid molecules which comprise an Mla nucleotide sequence derived from an Mla locus (e.g. Mla1, 6, 12) encoding an MLA polypeptide which is capable of recognising and activating a race specific defence response in a plant into which the nucleic acid is introduced and expressed, in response to challenge with a cognate Erysiphe graminis isolate. Also disclosed are novel methods for selecting such sequences based on the determination of an Mla (AT)n micro-satellite identified by the present inventors. Also provided is an novel 3 component activity assay, for assessing the ability of nucleic acid encoding a putative resistance (R) gene to confer resistance against a pathogen expressing a cognate Avr gene, which comprises the steps of: (a) selecting plant material which comprises plant cells which express a recessive gene conferring resistance against the pathogen, (b) introducing into the plant material, nucleic acid encoding (i) a detectable marker, (ii) a dominant susceptibility gene which inhibits the resistance conferred by the recessive gene, and (iii) the putative R gene, (c) challenging the plant material with the pathogen, (d) observing cells in the plant material in which the marker is expressed to determine the amount of pathogen growth present, and (e) correlating the amount of pathogen growth with the ability of the R gene to confer resistance against the pathogen. Also provided are corresponding methods and materials (e.g. vectors, polypeptides, plants, kits) based on the use of Mla nucleotide sequences or identification methods.
1 An isolated nucleic acid molecule which nucleic acid comprises an Mla nucleotide sequence derived from an Mla locus encoding an MLA polypeptide which is capable of recognising and activating a race specific defence response in a plant into which the nucleic acid is introduced and expressed, in response to challenge with a cognate Erysiphe graminis isolate.
2 A nucleic acid as claimed in claim 1 wherein the Mla locus is the Mla1 locus of Hordeum vulgare CI-16137 or the Mla6 locus of Hordeum vulgare CI-16151 or the Mla12 locus from Hordeum vulgare cultivar Sultan-5.
3 An isolated nucleic acid molecule which nucleic acid comprises an Mla nucleotide sequence which:
4 A nucleic acid as claimed in any one of claims 1 to 3 wherein the Mla nucleotide sequence is selected from a list consisting of:
5 A nucleic acid as claimed in claim 3 wherein the Mla nucleotide sequence encodes a derivative of an MLA resistance polypeptide shown in FIG. 10 or Annex V by way of addition, insertion, deletion or substitution of one or more amino acids.
6 A nucleic acid as claimed in any one of claims 1 to 3 wherein the Mla nucleotide sequence consists of an allelic, paralogous or orthologous variant of an Mla nucleotide sequence of FIG. 9 or FIG. 11.
7 An isolated nucleic acid which comprises a nucleotide sequence which is the complement of the Mla nucleotide sequence of any one of the preceding claims.
8 An isolated nucleic acid for use as a probe or primer, said nucleic acid consisting of a distinctive sequence of at least about 16-24 nucleotides in length, which sequence is (i) conserved between the Mla1 and Mla6 nucleotide sequences of FIG. 9, but not conserved with the other sequences shown therein, or conserved between at least two of the Mla1, Mla6 or Mla12 sequences of FIG. 11 (ii) a sequence degeneratively equivalent to said conserved sequence, or (iii) the complement sequence of either.
9 A nucleic acid primer as claimed in claim 8, selected from:
10 A method for isolating, identifying or locating a functional Mla allele, which method comprises the steps of:
11 A method as claimed in claim 10 wherein step (b) comprises the step of contacting nucleic acid in said preparation with a probe or primer adapted to identify the presence of an Mla (AT)n micro-satellite in the nucleic acid preparation.
12 A method as claimed in claim 11 wherein the Mla (AT)n micro-satellite sequence includes at least about 6, 8, 10, 12, 14, 16, 20, 24, 28, 32, 36, 40 or more AT repeats.
13 A method as claimed in claim 11 wherein the presence of the Mla (AT)n micro-satellite sequence is determined in a product amplified from the nucleic acid preparation.
14 A nucleic acid primer for use in the method of claim 13, selected from:
15 A method for identifying, cloning, or determining the presence within a plant of a nucleic acid as claimed in claim 2 or claim 6, which method employs a nucleic acid as claimed in claim 4, 8, 9 or 14.
16 A method as claimed in claim 15, which method comprises the steps of:
17 A method as claimed in claim 15, which method comprises the steps of:
18 A recombinant vector which comprises the nucleic acid of any one of claims 1 to 6.
19 A vector as claimed in claim 18 wherein the nucleic acid is operably linked to a promoter for transcription in a host cell, wherein the promoter is optionally an inducible promoter.
20 A vector as claimed in claim 18 or claim 19 which is a plant vector.
21 A method which comprises the step of introducing the vector of any one of claims 18 to 20 into a host cell, and optionally causing or allowing recombination between the vector and the host cell genome such as to transform the host cell.
22 A host cell containing or transformed with a heterologous vector of any one of claims 18 to 20.
23 A method for producing a transgenic plant, which method comprises the steps of:
24 A transgenic plant which is optionally selected from a species which is susceptible to powdery mildew, and which is obtainable by the method of claim 23, or which is a clone, or selfed or hybrid progeny or other descendant of said transgenic plant, which in each case includes a heterologous nucleic acid of any one of claims 1 to 6.
25 A part of propagule from a plant as claimed in claim 24, and which in either case includes a heterologous nucleic acid of any one of claims 1 to 6.
26 An isolated polypeptide which is encoded by the Mla nucleotide sequence of any one of claims 1 to 6.
27 A polypeptide as claimed in claim 26 which is an MLA resistance polypeptide shown in FIG. 5, FIG. 10 or Annex V.
28 A method of making the polypeptide of claim 26 or claim 27, which method comprises the step of causing or allowing expression from a nucleic acid of any one of claims 1 to 6 in a suitable host cell.
29 A polypeptide which comprises the antigen-binding site of an antibody having specific binding affinity for the polypeptide of claim 27.
30 A method for influencing or affecting the degree of resistance of a plant to a powdery mildew, which method comprises the step of causing or allowing expression of a heterologous nucleic acid as claimed in any one of claims 1 to 7 within the cells of the plant, following an earlier step of introducing the nucleic acid into a cell of the plant or an ancestor thereof.
31 A method as claimed in claim 30 for increasing a plant's powdery mildew disease resistance, wherein the nucleic acid is a nucleic acid as claimed in any one of claims 1 to 6.
32 A method as claimed in claim 31 which further comprises the step of manipulating a Rar1 and/or Rar2 gene in the plant.
33 An isolated nucleic acid molecule encoding the promoter or other UTR (3′ or 5′) of an Mla gene of claim 2, or a homologous variant thereof which has promoter activity.
34 A method for assessing the ability of nucleic acid encoding a putative resistance (R) gene to confer resistance against a pathogen expressing a cognate Avr gene, the method comprising the steps of:
35 A method as claimed in claim 34 wherein the amount of pathogen growth in step (d) is determined by comparison with cells in the plant material in which the marker is expressed and which have been challenged with pathogen but in which no pathogen growth is established.
36 A method as claimed in claim 34 or claim 35 further comprising step (f) correlating the number of cells in the plant material expressing the marker with the ability of the R gene to confer a hyper-sensitive resistance response against the pathogen.
37 A method as claimed in any one of claims 34 to 36 wherein the amount of pathogen for step (e) or the number of cells for step (f) is further compared against a corresponding control system in which either (1) no R gene is present, or (2) a corresponding pathogen not expressing a cognate AVR gene is used.
38 A method as claimed in any one of claims 34 to 37 wherein the nucleic acid introduced in step (b) is introduced as a first vector encoding (i) the detectable marker, (ii) the dominant susceptibility gene and a second vector encoding (iii) the putative R gene.
39 A method as claimed in claim 38 wherein the first and second vectors are co-introduced into the plant material such that they are at least transiently expressed therein.
40 A method as claimed in any one of claims 34 to 39 wherein the marker in step (b) is selected from:
41 A method as claimed in any one of claims 34 to 40 wherein the recessive gene of step (a) provides a broad resistance against the pathogen.
42 A method as claimed in claim 41 wherein the recessive gene of step (a) is the mlo gene, and the dominant susceptibility of (b) is the Mlo gene.
43 A method as claimed in any one of claims 34 to 42 wherein the R gene is selected from an Mla gene of claim 1, and the pathogen expressing the cognate Avr gene is the cognate Erysiphe graminis isolate.
44 A method as claimed in claim 43 wherein the R gene is selected from Mla6 and the isolate is A6, or the R gene is Mla1 and the isolate is k1
45 A method as claimed in any one of claims 34 to 44 for use in the identification of a putative pathogen expressing a cognate AVR gene for a selected R gene, or a putative inhibitor of the interaction between a selected pathogen expressing a cognate AVR gene and a selected R gene.
46 A plant vector for use in a method as claimed in any one of claims 34 to 45, which vector comprises:
47 A vector as claimed in claim 46 which is selected from:
48 A composition of matter comprising a first vector as claimed in claim 46 or claim 47;
49 A kit for assessing the ability of nucleic acid encoding a resistance (R) gene to confer resistance against a pathogen expressing a cognate Avr gene, the kit comprising:
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No related US applications are recorded in the indexed filing history.
Backward citations are indexed for grants only.
For biotic stress resistance, pathogen resistance, disease resistance: for fungal resistance
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: from plants
For detection or identification of organisms: for plants, fungi or algae
Oligonucleotides characterized by their use: plant traits
Term end not estimated
The present invention relates to methods and materials, particularly nucleic acids, for manipulating the resistance of plants to powdery mildew. It further relates to plants which have been modified using such methods and materials.
Genotype specific disease resistance in plants depends on the expression of complementary avirulence (Avr) genes in the pathogen and resistance (R) genes in the host. The final outcome of a matched R-Avr interaction is incompatibility i.e. containment of the pathogen at the site of penetration.
Numerous R genes have been cloned and characterized from a wide variety of plant species. Resistance genes that function in a gene-for-gene manner generally belong to one of four general classes based on motifs that are found within the encoded protein sequence. The first three classes include a cytoplasmic protein kinase, a protein with a cytoplasmic protein kinase and extracellular leucine rich repeats (LRRs) or proteins with LRRs that appear to be located extracellularly. Members of the fourth and largest class encode cytoplasmic proteins with a nucleotide-binding site (NBS) and several LRRs. Sequence diversity within the LRRs is thought to determine recognition specificity for proteins that are otherwise quite similar. The NBS-LRR class of resistance genes can be further subdivided into proteins with a coiled-coil or Toll-interleukin-1 receptor (TIR) homology domain at the amino terminus where they may have a function in directing certain protein-protein interactions.
FIG. 1: a three-component single-cell functional assay system based on Mlo, GFP, and Mla1, as described in Example 5.
FIG. 2: vector pUGLUM, a 9.8 kb plasmid harbouring both GFP and Mlo each driven by the ubiquitin 1 promoter, as described in Example 5.
FIGS. 3 and 4: two genes designated herein R gene A (Mla1 Gene Sequence) and B (Mla1 Gene Homologue Sequence) obtained from cosmid p6-49-2-15 and p6-49-2-7 The genes showing significant sequence-relatedness to NBS-LRR type R genes. The bold letters represent exon sequences.
Table 1: 12 cosmids isolated from the library representing genomic DNA from cultivar AlgR Mla1 (see Example 4).
Table 2: testing for the presence of Mla1 in cosmid clones—results obtained upon transfection of pUGLUM only, pUGLUM co-bombarded with cosmid p7-35-2, and cosmid p6-49-2.
Table 3: testing the function of R genes A and B separately by transient expression in detached leaves by co-bombardment of each subclone together with pUGLUM. A 15 kb EcoRI subclone containing only R gene A is designated p6-49-2-15. A 7 kb DraI subclone containing only R gene B is designated p6-49-2-7.