Patent Application
20230257765
The present invention relates to a transgenic plant which has integrated into its genome an exogenous polynucleotide encoding a polypeptide which confers resistance to one or more races of Puccinia graminis, such as the Ug99 group of races of Puccinia graminis f. sp. tritici.
A Sequence Listing is provided herewith as a Sequence Listing XML, “522819US01 Stem rust resistance gene” created on May 4, 2023 and having a size of 100 KB. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
The fungal pathogen Puccinia graminis Pers. f. sp. tritici (Pgt), the causal agent of wheat stem rust, is a major threat to wheat production, in part because of the level of devastation it can inflict on wheat crops with near complete losses in severe epidemics. Although rust resistant cultivars had controlled the disease for over 40 years, the appearance in Uganda in 1999 of Pgt race TTKSK, commonly known as Ug99, and the subsequent spread of its derivatives to other parts of Africa and the Middle East, has highlighted the continuing danger of stem rust. Ug99 and derivative strains are virulent on plants containing the stem rust resistance gene Sr31, which had previously provided effective resistance for over thirty years (Ellis et al., 2014), as well as many other common stem rust resistance (Sr) genes (Singh et al., 2011). The recent appearance of another Pgt race (different to the Ug99 lineage) in Germany and Ethiopia in 2013-2014 attacking important commercial wheat varieties has raised further serious concerns for wheat production. While an international collaboration called the Borlaug Global Rust Initiative (BGRI) has since been actively involved in strategies to control stem rust, changes in global climate and warming can potentially open additional areas for rust epidemics which were previously considered safe from stem rust (Chakraborty et al., 2011).
The control of wheat rust is dependent on the incorporation of effective resistance genes during breeding and combinations of multiple stem rust resistance genes are crucial for providing durable resistance, which necessitates the identification of new resistance genes. Wild and cultivated relatives of wheat provide an important pool of new genes effective against wheat rust pathogens. Cereal rye (Secale cereale) is one such source and several rye genes have been used in breeding rust resistant bread wheat and triticale. Indeed, the Sr31 resistance gene was introgressed from rye as a full chromosome substitution for wheat chromosome 1B or as a translocation of the short arm of rye chromosome 1 (1RS) from the rye cultivar Petkus (Zeller, 1973) to the long arm of wheat chromosome 1B (1BL) and conferred stem rust resistance for over 30 years in the field (Ellis et al., 2014). Sr50 (previously known as SrR) was also introgressed into wheat as translocations of 1RS to the long arms of wheat chromosomes 1B and 1D, but sourced from the rye cultivar Imperial (Shepherd 1973; Mago et al., 2004). A third stem rust resistance gene, also known as Sr1RSAmigo, was introgressed as a 1RS translocation from rye cultivar Insave to wheat chromosome 1A in cultivar Amigo (Zeller and Fuchs, 1983). Because these rye genes gave resistance to all known Pgt strains, it was not clear whether they represented different resistance specificities. Both Sr31 and Sr50 are associated with a cluster of genes on 1RS (Mago et al., 2004 and 2005), encoding coiled-coil nucleotide binding leucine-rich repeat (CC-NB-LRR) proteins orthologous to the barley Mla gene cluster, that provides resistance to Blumeria graminis f. sp. hordei (powdery mildew pathogen) (Wei et al., 1999 and 2002). However, it was not known which if any of these proteins was the product of the Sr50 gene itself. The recently cloned Sr33 gene from Triticum tauschii (Periyannan et al., 2013) is an ortholog of Mla, and like Sr50 provides resistance to worldwide Pgt isolates so again it has not been possible to distinguish these two specificities.
The Mla locus of barley is one of the most diverse classes of resistance genes in cereals, encoding more than 30 different alleles with different resistance specificities to barley powdery mildew (Seeholzer et al., 2010). This locus is also a potential source of useful disease resistance in other cereals, including wheat. For instance, the wheat TmMla1 gene present in the diploid A-genome wheat species Triticum monococcum is an ortholog of Mla and confers race-specific resistance to wheat powdery mildew (Jordan et al., 2011). The recently identified Sr33 gene, transferred to wheat from the diploid D-genome species Ae. tauschii, is the first known member of the Mla family to provide resistance to Pgt (Periyannan et al., 2013).
There is an urgent need for the identification of genes which confer at least some level of resistance to plants, especially wheat, against Puccinia graminis, such as the Ug99 group of races of Puccinia graminis f. sp. tritici.
The present inventors have identified polypeptides which confer at least some level of resistance to plants, especially rye and wheat, against Puccinia graminis, such as the Ug99 group of races of Puccinia graminis f. sp. tritici.
Thus, in a first aspect the present invention provides a transgenic plant which has integrated into its genome an exogenous polynucleotide encoding a polypeptide which confers resistance to Puccinia graminis, wherein the polynucleotide is operably linked to a promoter capable of directing expression of the polynucleotide in a cell of the plant.
In an embodiment, the Puccinia graminis is Puccinia graminis f. sp. tritici. In a further embodiment, the Puccinia graminis f. sp. tritici is a race of the Ug99 group.
In another embodiment, the transgenic plant has enhanced resistance to Puccinia graminis when compared to an isogenic plant lacking the exogenous polynucleotide.
In an embodiment, the polypeptide is an Sr50 polypeptide.
In a further embodiment,
i) the polypeptide comprises amino acids having a sequence as provided in SEQ ID NO:1, a biologically active fragment thereof, or an amino acid sequence which is at least 82% identical to SEQ ID NO:1, and/or
ii) the polynucleotide comprises nucleotides having a sequence as provided in SEQ ID NO:10, a sequence which is at least 82% identical to SEQ ID NO:10, or a sequence which hybridizes to SEQ ID NO:10.
In an embodiment, the polypeptide comprises one or more, preferably all, of a coiled coil (CC) domain, an nucleotide binding (NB) domain and a leucine rich repeat (LRR) domain.
In a further embodiment, the polypeptide comprises one or more, preferably all, of a p-loop motif, and a kinase3a motif in the NB domain.
In an embodiment, the p-loop motif comprises the sequence GxxGxGK(T/S)T (SEQ ID NO:4), more preferably the sequence GFGGLGKTT (SEQ ID NO:5).
In an embodiment, the kinase 3a motif comprises the sequence GxxxxxTxR (SEQ ID NO:6), more preferably the sequence GSRLITTTR (SEQ ID NO:7).
In a further embodiment, the LRR domain comprises about 5 to about 20, or about 10 to about 20, imperfect repeats of the sequence xxLxLxxxx (SEQ ID NO:8).
In an embodiment, the polypeptide confers greater resistance to Puccinia graminis f. sp. tritici race TTKSK than Sr33 (with a sequence of amino acids as provided in SEQ ID NO:13). In another embodiment, Sr33 (with a sequence of amino acids as provided in SEQ ID NO:13) confers greater resistance to Puccinia graminis f. sp. tritici race QFCSC than a polypeptide of the invention. In an embodiment, as detailed in Example 2, the greater resistance is determined when the polypeptide of the invention is in T. aestivum line Gabo 1DL.1RS-DR.A1 and when Sr33 is in T. aestivum line Westonia/CS1D5405.
Preferably, the plant is a cereal plant. Examples of transgenic cereal plants of the invention include, but are not limited to wheat, barley, maize, rice, oats, sorghum and triticale. In a particularly preferred embodiment, the plant is wheat.
In a further embodiment, the plant comprises one or more further exogenous polynucleotides encoding another plant pathogen resistance polypeptide. Examples of such other plant pathogen resistance polypeptides include, but are not limited to, Lr34, Lr1, Lr3, Lr2a, Lr3ka, Lr11, Lr13, Lr16, Lr17, Lr18, Lr21, LrB, Sr35 and Sr33. In an embodiment, the plant at least further comprises an exogenous polynucleotide encoding plant pathogen resistance polypeptide Sr33. For example, the at least further comprises an exogenous polynucleotide encoding a polypeptide comprising amino acids having a sequence as provided in SEQ ID NO:13 or SEQ ID NO:14, or an amino acid sequence which is at least 87% identical, at least 90% identical, or at least 95% identical, to one or both of SEQ ID NO:13 and SEQ ID NO:14 which confers resistance to Puccinia graminis.
Preferably, the plant is homozygous for the exogenous polynucleotide.
In an embodiment, the plant is growing in a field.
In a further aspect, the present invention provides a transgenic plant which has integrated into its genome an exogenous polynucleotide encoding a polypeptide which comprises amino acids having a sequence as provided in SEQ ID NO:1, or an amino acid sequence which is at least 82% identical to SEQ ID NO:1, wherein the polynucleotide is operably linked to a promoter capable of directing expression of the polynucleotide in a cell of the plant.
Also provided is a population of at least 100 transgenic plants of the invention growing in a field.
In a further aspect, the present invention provides a process for identifying a polynucleotide encoding a polypeptide which confers resistance to Puccinia graminis comprising:
i) obtaining a polynucleotide operably linked to a promoter, the polynucleotide encoding a polypeptide comprising amino acids having a sequence as provided in SEQ ID NO:1, a biologically active fragment thereof, or an amino acid sequence which is at least 82% identical to SEQ ID NO:1,
ii) introducing the polynucleotide into a plant,
iii) determining whether the level of resistance to Puccinia graminis is modified relative to an isogenic plant lacking the polynucleotide, and
iv) optionally, selecting a polynucleotide which when expressed confers resistance to Puccinia graminis.
In an embodiment the process has one or more of the following,
a) the polynucleotide comprises nucleotides having a sequence as provided in SEQ ID NO:10, a sequence which is at least 82% identical to SEQ ID NO:10, or a sequence which hybridizes to SEQ ID NO:10,
b) the plant is a cereal plant such as a wheat plant,
c) the polypeptide is a plant polypeptide or mutant thereof, and
d) step ii) further comprises stably integrating the polynucleotide operably linked to a promoter into the genome of the plant.
Also provided is a substantially purified and/or recombinant Puccinia graminis plant resistance polypeptide.
In an embodiment, the polypeptide is an Sr50 polypeptide.
In another embodiment, the polypeptide comprises amino acids having a sequence as provided in SEQ ID NO:1, a biologically active fragment thereof, or an amino acid sequence which is at least 82% identical, at least 90% identical, or at least 95% identical, to SEQ ID NO:1.
In a further aspect, the present invention provides a substantially purified and/or recombinant polypeptide comprising amino acids having a sequence as provided in SEQ ID NO:1, or an amino acid sequence which is at least 82% identical, at least 90% identical, or at least 95% identical, to SEQ ID NO:1.
In an embodiment, a polypeptide of the invention is a fusion protein further comprising at least one other polypeptide sequence. The at least one other polypeptide may be, for example, a polypeptide that enhances the stability of a polypeptide of the present invention, or a polypeptide that assists in the purification or detection of the fusion protein.
In yet a further aspect, the present invention provides an isolated and/or exogenous polynucleotide comprising nucleotides having a sequence as provided in SEQ ID NO:10, a sequence which is at least 82% identical to SEQ ID NO:10, a sequence encoding a polypeptide of the invention, or a sequence which hybridizes to SEQ ID NO:10.
In another aspect, the present invention provides a chimeric vector comprising the polynucleotide of the invention.
Preferably, the polynucleotide is operably linked to a promoter.
In a further aspect, the present invention provides a recombinant cell comprising an exogenous polynucleotide of the invention and/or a vector of the invention.
The cell can be any cell type such as, but not limited to, a plant cell, a bacterial cell, an animal cell or a yeast cell.
Preferably, the cell is a plant cell. More preferably, the plant cell is a cereal plant cell. Even more preferably, the cereal plant cell is a wheat cell.
In a further aspect, the present invention provides a method of producing the polypeptide of the invention, the method comprising expressing in a cell or cell free expression system the polynucleotide of the invention.
Preferably, the method further comprises isolating the polypeptide.
In yet another aspect, the present invention provides a transgenic non-human organism comprising an exogenous polynucleotide of the invention, a vector of the invention and/or a recombinant cell of the invention.
Preferably, the transgenic non-human organism is a plant. Preferably, the plant is a cereal plant. More preferably, the cereal plant is a wheat plant.
In another aspect, the present invention provides a method of producing the cell of the invention, the method comprising the step of introducing the polynucleotide of the invention, or a vector of the invention, into a cell.
Preferably, the cell is a plant cell.
In a further aspect, the present invention provides a method of producing a transgenic plant of the invention, the method comprising the steps of
i) introducing a polynucleotide of the invention and/or a vector of the invention into a cell of a plant,
ii) regenerating a transgenic plant from the cell, and
iii) optionally harvesting seed from the plant, and/or
iv) optionally producing one or more progeny plants from the transgenic plant, thereby producing the transgenic plant.
In a further aspect, the present invention provides a method of producing a plant which has integrated into its genome a polynucleotide encoding a polypeptide which confers resistance to Puccinia graminis, the method comprising the steps of
i) crossing two parental plants, wherein at least one plant comprises a polynucleotide encoding a polypeptide which confers resistance to Puccinia graminis,
ii) screening one or more progeny plants from the cross for the presence or absence of the polynucleotide, and
iii) selecting a progeny plant which comprise the polynucleotide, thereby producing the plant.
In an embodiment, at least one of the parental plants is a transgenic plant of the invention, and the selected progeny plant comprises an exogenous polynucleotide encoding a polypeptide which confers resistance to Puccinia graminis.
In a further embodiment, at least one of the parental plants is a tetraploid or hexaploid wheat plant.
In yet another embodiment, step ii) comprises analysing a sample comprising DNA from the plant for the polynucleotide.
In another embodiment, step iii) comprises
i) selecting progeny plants which are homozygous for the polynucleotide, and/or
ii) analysing the plant or one or more progeny plants thereof for resistance to Puccinia graminis.
In an embodiment, the method further comprises
iv) backcrossing the progeny of the cross of step i) with plants of the same genotype as a first parent plant which lacked a polynucleotide encoding a polypeptide which confers resistance to Puccinia graminis for a sufficient number of times to produce a plant with a majority of the genotype of the first parent but comprising the polynucleotide, and
iv) selecting a progeny plant which has resistance to Puccinia graminis.
In yet another aspect, a method of the invention further comprises the step of analysing the plant for at least one other genetic marker.
Also provide is a plant produced using a method of the invention.
In another aspect, the present invention provides for the use of the polynucleotide of the invention, or a vector of the invention, to produce a recombinant cell and/or a transgenic plant.
In an embodiment, the transgenic plant has enhanced resistance to Puccinia graminis when compared to an isogenic plant lacking the exogenous polynucleotide and/or vector.
In a further aspect, the present invention provides a method for identifying a plant comprising a polynucleotide encoding a polypeptide which confers resistance to Puccinia graminis, the method comprising the steps of
i) obtaining a nucleic acid sample from a plant, and
ii) screening the sample for the presence or absence of the polynucleotide, wherein presence of the polynucleotide indicates that the plant is resistant to Puccinia graminis.
In an embodiment, the polynucleotide encodes a polypeptide of the invention.
In a further embodiment, the method identifies a transgenic plant of the invention.
In another embodiment, the method further comprises producing a plant from a seed before step i).
Also provided is a plant part of the plant of the invention.
In an embodiment, the plant part is a seed that comprises an exogenous polynucleotide which encodes a polypeptide which confers resistance to Puccinia graminis.
In a further aspect, the present invention provides a method of producing a plant part, the method comprising,
a) growing a plant of the invention, and
b) harvesting the plant part.
In another aspect, the present invention provides a method of producing flour, wholemeal, starch or other product obtained from seed, the method comprising;
a) obtaining seed of the invention, and
b) extracting the flour, wholemeal, starch or other product.
In a further aspect, the present invention provides a product produced from a plant of the invention and/or a plant part of the invention.
In an embodiment, the part is a seed.
In an embodiment, the product is a food product or beverage product. Examples include, but are not limited to;
i) the food product being selected from the group consisting of: flour, starch, leavened or unleavened breads, pasta, noodles, animal fodder, breakfast cereals, snack foods, cakes, malt, beer, pastries and foods containing flour-based sauces, or
ii) the beverage product being beer or malt.
In an alternative embodiment, the product is a non-food product. Examples include, but are not limited to, films, coatings, adhesives, building materials and packaging materials.
In a further aspect, the present invention provides a method of preparing a food product of the invention, the method comprising mixing seed, or flour, wholemeal or starch from the seed, with another food ingredient.
In another aspect, the present invention provides a method of preparing malt, comprising the step of germinating seed of the invention.
Also provided is the use of a plant of the invention, or part thereof, as animal feed, or to produce feed for animal consumption or food for human consumption.
In a further aspect, the present invention provides a composition comprising one or more of a polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, or a recombinant cell of the invention, and one or more acceptable carriers.
In another aspect, the present invention provides a method of identifying a compound that binds to a polypeptide comprising amino acids having a sequence as provided in SEQ ID NO:1, a biologically active fragment thereof, or an amino acid sequence which is at least 82% identical to SEQ ID NO:1, the method comprising:
i) contacting the polypeptide with a candidate compound, and
ii) determining whether the compound binds the polypeptide.
Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.
SEQ ID NO:1—Amino acid sequence of stem rust resistance polypeptide (Sr50).
SEQ ID NO:2—Amino acid sequence of Sr50 variant from Svalofs Otello and Frontier.
SEQ ID NO:3—Amino acid sequence of Sr50 variant from Dwarf Petkus.
SEQ ID NO:4—Consenus p-loop motif.
SEQ ID NO:5—P-loop motif of polypeptide provided as SEQ ID NO:1.
SEQ ID NO:6—Consenus kinase 3a motif.
SEQ ID NO:7—Kinase 3a motif of polypeptide provided as SEQ ID NO:1.
SEQ ID NO:8—Consensus repeat of the LRR domain.
SEQ ID NO:9—Nucleotide sequence of cDNA encoding stem rust resistance polypeptide (Sr50).
SEQ ID NO:10—Nucleotide sequence of open reading frame encoding stem rust resistance polypeptide (Sr50).
SEQ ID NO:11—Nucleotide sequence of pVecNeoSr50 expression construct.
SEQ ID NO:12—Nucleotide sequence of pVecBarSr50 expression construct.
SEQ ID NO:13—Amino acid sequence of stem rust resistance polypeptide (from haplotype I) (Sr33).
SEQ ID NO:14—Amino acid sequence of allelic variant of the stem rust resistance polypeptide provided as SEQ ID NO:13 (from haplotype II) (Sr33).
SEQ ID NO:15—Functional nuclear export signal NES from HIV Rev.
SEQ ID NOs 17 to 59—Oligonucleotide primers.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, plant molecular biology, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
As used herein, the term about, unless stated to the contrary, refers to +/−10%, more preferably +/−5%, more preferably +/−1%, more preferably +/−0.5%, of the designated value.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
As used herein, “stem rust” refers to the disease of plants caused by Puccinia graminis or to the causative fungal pathogen, Puccinia graminis, as the context determines. As used herein, “wheat stem rust” refers to the disease of plants caused by Puccinia graminis f. sp. tritici or to the causative fungal pathogen, Puccinia graminis f. sp. tritici, as the context determines.
The Ug99 group of races of wheat stem rust (Puccinia graminis f. sp. tritici) (also known as ‘TTKSK’ under the North American nomenclature system) is a well known fungal pathogen of wheat and is commonly present in wheat fields in countries such as in Africa and the Middle East (Singh et al., 2011; Hodson et al., 2012). Ug99 can cause major crop losses and is virulent against resistance genes that have previously protected wheat against stem rust. There are currently eight known variants of group Ug99 which are closely related based on DNA marker analysis. Each variant of the pathogen which differs in its virulence/avirulence profile on a panel of wheat plants each comprising a different resistance R gene is known as a “race” of the pathogen. The Ug99 group of isolates are all closely related and are believed to have evolved from a common ancestor, but may differ in their virulence/avirulence profiles in which case they are considered different races. Seven of these eight variants are summarized in Table 2 of Singh et al. (2011). In an embodiment, the Ug99 group of stem rust races exhibit virulence on wheat plants comprising one or more of the resistance genes Sr31, Sr21, Sr24 and Sr36 (Singh et al., 2011). In one embodiment, the Ug99 group of stem rust races of Puccinia graminis f. sp. tritici has virulence at least to wheat plants comprising the resistance gene Sr31 (Pretorius et al., 2000).
The present invention relates to polypeptides which confer resistance to a plant, for example a wheat plant, to stem rust, preferably to wheat stem rust such as the Ug99 group of races. In a preferred embodiment, the polypeptide is encoded by an allele or variant of an Sr50 gene which confers resistance to wheat stem rust. Examples of such polypeptides include, but are not limited to, those comprising an amino acid sequence as provided in SEQ ID NO:1. The polypeptide of the invention confers enhanced resistance to stem rust, preferably wheat stem rust such as the Ug99 group of races of Puccinia graminis f. sp. tritici when compared to an isogenic plant lacking a gene encoding the polypeptide. This term also refers to the naturally produced protein (or wild-type protein from which a mutant protein is derived) encoded by a gene conferring upon a plant (for example, wheat), when grown in normal field conditions, enhanced resistance to stem rust such as the Ug99 group of races of Puccinia graminis f. sp. tritici. In a preferred embodiment, the polypeptide of the invention confers resistance specifically to stem rust, preferably specifically to wheat stem rust, more preferably it does not confer resistance to wheat leaf rust caused by the fungal pathogen Puccinia triticina and/or to powdery mildew. In this context, “specifically to stem rust” and “specifically to wheat stem rust” means that the conferred resistance is preferentially to stem rust or wheat stem rust in comparison to another fungal pathogen of the same plant species, preferably to many or most other fungal pathogens of the same species. In a more preferred embodiment, the polypeptide of the invention confers resistance to stem rust and at least two, or all three, of leaf rust, stripe rust and powdery mildew, preferably in wheat. In an embodiment, polypeptides of the invention are not encoded by the Sr35 gene of a wheat plant. In an embodiment, polypeptides of the invention are not encoded by the Sr35 gene of a wheat plant or its homologs, such as those that are at least 50% identical in amino acid sequence to the Sr35 polypeptide. In another embodiment, polypeptides of the invention are not encoded by the Sr33 gene of a wheat plant. In an embodiment, polypeptides of the invention are not encoded by the Sr33 gene of a wheat plant or its homologs, such as those that are at least 87% identical in amino acid sequence to the Sr33 polypeptide as described in WO 2014/000594 (SEQ ID NO's 13 and 14 herein). Thus, in an embodiment, a polypeptide of the invention does not comprise amino acids having a sequence at least 87% identical in amino acid sequence to SEQ ID NO:13 and/or SEQ ID NO:14. In a further embodiment, a polypeptide of the invention has an amino acid sequence which is more closely related (has a higher % identity level) to SEQ ID NO:1 than SEQ ID NO:13 and/or 14.
In a further embodiment, when expressed in a transgenic plant infected with stem rust, such as with a Ug99 race of Puccinia graminis f. sp. tritici, the cells of the plant display little, if any, signs of cell death (autofluorescence), for instance when compared to an isogenic plant expressing Sr45.
Polypeptides of the invention typically comprise a coiled coil (CC) domain towards the N-terminus, followed by an nucleotide binding (NB) domain and a leucine rich repeat (LRR) domain towards the C-terminus (see
A coiled-coil domain or motif is a structural motif which is one of the most common tertiary structures of proteins where a-helices are coiled together like the strands of a rope. Computer programs have been devised to detect heptads and resulting in coiled-coil structures (see, for example Delorenzi and Speed, 2002). Coiled coils typically comprise a repeated pattern, hxxhcxc, of hydrophobic (h) and charged (c) amino-acid residues, referred to as a heptad repeats. The positions in the heptad repeat are usually labeled abcdefg, where a and d are the hydrophobic positions, often being occupied by isoleucine, alanine, leucine or valine. Folding a protein with these hepatds into an a-helical secondary structure causes the hydrophobic residues to be presented as a ‘stripe’ that coils gently around the helix in left-handed fashion, forming an amphipathic structure.
The NB domain is present in resistance genes as well as several kinases such as ATP/GTP-binding proteins. This domain typically contains three motifs: kinase-la (p-loop), a kinase-2, and a putative kinase-3a (Traut 1994; Tameling et al., 2002). The consensus sequence of GxxGxGK(T/S)T (SEQ ID NO:4) (GFGGLGKTT (SEQ ID NO:5) in the polypeptide which confers resistance to Puccinia graminis provided as SEQ ID NO:1), and GxxxxxTxR (SEQ ID NO:6) (GSRLITTTR (SEQ ID NO:7) in the polypeptide which confers resistance to Puccinia graminis provided as SEQ ID NO:1) for the resistance gene motifs p-loop, kinase-2, and the putative kinase-3a, respectively, are different from those present in other NB-encoding proteins. Other motifs present in the NB domain of NB/LRR-type resistance genes are GLPL, RNBS-D and MHD (Meyers et al., 1999). The sequences interspersing these motifs and domains can be very different even among homologues of a resistance gene (Michelmore and Meyers, 1998; Pan et al., 2000).
A leucine-rich domain is a protein structural motif that forms an α/β horseshoe fold (Enkhbayar et al., 2004). The LRR domain contains 9-41 imperfect repeats, each about 25 amino acids long with a consensus amino acid sequence of xxLxLxxxx (SEQ ID NO:8) (Cooley et al., 2000). In an embodiment, a polypeptide of the invention comprises about 10 to about 20, more preferably about 12 to about 18, more preferably about 15 leucine rich repeats. These repeats commonly fold together to form a solenoid protein domain Typically, each repeat unit has beta strand-turn-alpha helix structure, and the assembled domain, composed of many such repeats, has a horseshoe shape with an interior parallel beta sheet and an exterior array of helices.
In an embodiment, the polypeptide comprises one, two, three, four or more amino acids which are present in the amino acid sequence provided as SEQ ID NO:1 but which are not found in the corresponding amino acid position of a polypeptide comprising amino acids having a sequence as provided in SEQ ID NO:13 and SEQ ID NO:14.
In an embodiment, the polypeptide does not comprise amino acids having a sequence as provided in SEQ ID NO:1 or SEQ ID NO:2, a biologically active fragment thereof, or an amino acid sequence which is at least 87% identical to one or both of SEQ ID NO:13 and SEQ ID NO:14. In an embodiment, polypeptide does not comprise amino acids having a sequence as provided in SEQ ID NO:13 or SEQ ID NO:14.
In an embodiment, the polypeptide is from S. cereale.
As used herein, “resistance” is a relative term in that the presence of a polypeptide of the invention (i) reduces the disease symptoms of a plant comprising the gene (R gene) that confers resistance, relative to a plant lacking the R gene, and/or (ii) reduces pathogen reproduction or spread on a plant comprising the R gene. Resistance as used herein is relative to the “susceptible” response of a plant to the same pathogen. Typically, the presence of the R gene improves at least one production trait of a plant comprising the R gene when infected with the pathogen, such as grain yield, when compared to an isogenic plant infected with the pathogen but lacking the R gene. The isogenic plant may have some level of resistance to the pathogen, or may be classified as susceptible. Thus, the terms “resistance” and “enhanced resistance” are generally used herein interchangeably. Furthermore, a polypeptide of the invention does not necessarily confer complete pathogen resistance, for example when some symptoms still occur or there is some pathogen reproduction on infection but at a reduced amount Enhanced resistance can be determined by a number of methods known in the art such as analysing the plants for the amount of pathogen and/or analysing plant growth or the amount of damage or disease symptoms to a plant in the presence of the pathogen, and comparing one or more of these parameters to an isogenic plant lacking an exogenous gene encoding a polypeptide of the invention.
By “substantially purified polypeptide” or “purified polypeptide” we mean a polypeptide that has generally been separated from the lipids, nucleic acids, other peptides, and other contaminating molecules with which it is associated in its native state. Preferably, the substantially purified polypeptide is at least 90% free from other components with which it is naturally associated.
Transgenic plants and host cells of the invention may comprise an exogenous polynucleotide encoding a polypeptide of the invention. In these instances, the plants and cells produce a recombinant polypeptide. The term “recombinant” in the context of a polypeptide refers to the polypeptide encoded by an exogenous polynucleotide when produced by a cell, which polynucleotide has been introduced into the cell or a progenitor cell by recombinant DNA or RNA techniques such as, for example, transformation. Typically, the cell comprises a non-endogenous gene that causes an altered amount of the polypeptide to be produced. In an embodiment, a “recombinant polypeptide” is a polypeptide made by the expression of an exogenous (recombinant) polynucleotide in a plant cell.
The terms “polypeptide” and “protein” are generally used interchangeably.
The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 150 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 150 amino acids. More preferably, the query sequence is at least 500 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 500 amino acids. More preferably, the query sequence is at least 750 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 750 amino acids. Even more preferably, the query sequence is at least 900 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 900 amino acids. Even more preferably, the GAP analysis aligns two sequences over their entire length.
As used herein a “biologically active” fragment is a portion of a polypeptide of the invention which maintains a defined activity of the full-length polypeptide such as when expressed in a plant, such as wheat, confers (enhanced) resistance to stem rust, preferably wheat stem rust such as the Ug99 group of races of Puccinia graminis f. sp. tritici when compared to an isogenic plant not expressing the polypeptide. Biologically active fragments can be any size as long as they maintain the defined activity but are preferably at least 750 or at least 900 amino acid residues long. Preferably, the biologically active fragment maintains at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the full length protein.
With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
Amino acid sequence mutants of the polypeptides of the present invention can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the present invention, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired characteristics. Preferred amino acid sequence mutants have only one, two, three, four or less than 10 amino acid changes relative to the reference wildtype polypeptide.
Mutant (altered) polypeptides can be prepared using any technique known in the art, for example, using directed evolution or rational design strategies (see below). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they confer resistance to Puccinia graminis (for example, a race of the Ug99 group of Puccinia graminis f sp. tritici) such as by producing a transgenic plant expressing the mutated/altered DNA and determining the ability of the plant to produce grain in the presence of the pathogen.
In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. In order to maintain activity, sites of interest include those not in an active site, such as a CC, BD or LRR domain, and those which are not highly conserved between different species. These sites, especially those falling within a sequence of at least three other non-conserved sites can generally be substituted in a relatively conservative or non-conservative manner Examples of conservative substitutions are shown in Table 1 under the heading of “exemplary substitutions”.
In a preferred embodiment a mutant/variant polypeptide has one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 1. In a preferred embodiment, the changes are not in one or more of the motifs which are highly conserved between the different polypeptides provided herewith. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell.
In an embodiment, the protein of the invention is a CC-NB-LRR plant pathogen resistance gene which comprises domains configured as shown in
The primary amino acid sequence of a polypeptide of the invention can be used to design variants/mutants thereof based on comparisons with closely related resistance polypeptides comprising NB and LRR domains, more preferably CC, NB and LRR domains. As the skilled addressee will appreciate, residues highly conserved amongst closely related CC-NB-LRR proteins are less likely to be able to be altered, especially with non-conservative substitutions, and activity maintained than less conserved residues (see above).
Also included within the scope of the invention are polypeptides of the present invention which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. The polypeptides may be post-translationally modified in a cell, for example by phosphorylation, which may modulate its activity. These modifications may serve to increase the stability and/or bioactivity of the polypeptide of the invention.
In directed evolution, random mutagenesis is applied to a protein, and a selection regime is used to pick out variants that have the desired qualities, for example, increased activity. Further rounds of mutation and selection are then applied. A typical directed evolution strategy involves three steps:
1) Diversification: The gene encoding the protein of interest is mutated and/or recombined at random to create a large library of gene variants. Variant gene libraries can be constructed through error prone PCR (see, for example, Leung, 1989, Cadwell and Joyce, 1992), from pools of DNasel digested fragments prepared from parental templates (Stemmer, 1994a; Stemmer, 1994b: Crameri et al., 1998; Coco et al., 2001) from degenerate oligonucleotides (Ness et al., 2002, Coco, 2002) or from mixtures of both, or even from undigested parental templates (Zhao et al., 1.998; Eggert et al., 2005; Jézéquek et al, 2008) and are usually assembled through PCR. Libraries can also be made from parental sequences recombined in vivo or in vitro by either homologous or non-homologous recombination (Ostermeier et al., 1999; Volkov et al., 1999; Sieber et al., 2001). Variant gene libraries can also be constructed by sub-cloning a gene of interest into a suitable vector, transforming the vector into a “mutator” strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. Variant gene libraries can also be constructed by subjecting the gene of interest to DNA shuffling (i.e., in vitro homologous recombination of pools of selected mutant genes by random fragmentation and reassembly) as broadly described by Harayama (1998).
2) Selection: The library is tested for the presence of mutants (variants) possessing the desired property using a screen or selection. Screens enable the identification and isolation of high-performing mutants by hand, while selections automatically eliminate all nonfunctional mutants. A screen may involve screening for the presence of known conserved amino acid motifs. Alternatively, or in addition, a screen may involve expressing the mutated polynucleotide in a host organism or part thereof and assaying the level of activity.
3) Amplification: The variants identified in the selection or screen are replicated many fold, enabling researchers to sequence their DNA in order to understand what mutations have occurred.
Together, these three steps are termed a “round” of directed evolution. Most experiments will entail more than one round. In these experiments, the “winners” of the previous round are diversified in the next round to create a new library. At the end of the experiment, all evolved protein or polynucleotide mutants are characterized using biochemical methods.
A protein can be designed rationally, on the basis of known information about protein structure and folding. This can be accomplished by design from scratch (de novo design) or by redesign based on native scaffolds (see, for example, Hellinga, 1997; and Lu and Berry, Protein Structure Design and Engineering, Handbook of Proteins 2, 1153-1157 (2007)). Protein design typically involves identifying sequences that fold into a given or target structure and can be accomplished using computer models. Computational protein design algorithms search the sequence-conformation space for sequences that are low in energy when folded to the target structure. Computational protein design algorithms use models of protein energetics to evaluate how mutations would affect a protein's structure and function. These energy functions typically include a combination of molecular mechanics, statistical (i.e. knowledge-based), and other empirical terms. Suitable available software includes IPRO (Interative Protein Redesign and Optimization), EGAD (A Genetic Algorithm for Protein Design), Rosetta Design, Sharpen, and Abalone.
The present invention refers to various polynucleotides. As used herein, a “polynucleotide” or “nucleic acid” or “nucleic acid molecule” means a polymer of nucleotides, which may be DNA or RNA or a combination thereof, and includes genomic DNA, mRNA, cRNA, and cDNA. Less preferred polynucleotides include tRNA, siRNA, shRNA and hpRNA. It may be DNA or RNA of cellular, genomic or synthetic origin, for example made on an automated synthesizer, and may be combined with carbohydrate, lipids, protein or other materials, labelled with fluorescent or other groups, or attached to a solid support to perform a particular activity defined herein, or comprise one or more modified nucleotides not found in nature, well known to those skilled in the art. The polymer may be single-stranded, essentially double-stranded or partly double-stranded. Basepairing as used herein refers to standard basepairing between nucleotides, including G:U basepairs. “Complementary” means two polynucleotides are capable of basepairing (hybridizing) along part of their lengths, or along the full length of one or both. A “hybridized polynucleotide” means the polynucleotide is actually basepaired to its complement. The term “polynucleotide” is used interchangeably herein with the term “nucleic acid”. Preferred polynucleotides of the invention encode a polypeptide of the invention.
By “isolated polynucleotide” we mean a polynucleotide which has generally been separated from the polynucleotide sequences with which it is associated or linked in its native state, if the polynucleotide is found in nature. Preferably, the isolated polynucleotide is at least 90% free from other components with which it is naturally associated, if it is found in nature. Preferably the polynucleotide is not naturally occurring, for example by covalently joining two shorter polynucleotide sequences in a manner not found in nature (chimeric polynucleotide).
The present invention involves modification of gene activity and the construction and use of chimeric genes. As used herein, the term “gene” includes any deoxyribonucleotide sequence which includes a protein coding region or which is transcribed in a cell but not translated, as well as associated non-coding and regulatory regions. Such associated regions are typically located adjacent to the coding region or the transcribed region on both the 5′ and 3′ ends for a distance of about 2 kb on either side. In this regard, the gene may include control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals in which case the gene is referred to as a “chimeric gene”. The sequences which are located 5′ of the coding region and which are present on the mRNA are referred to as 5′ non-translated sequences. The sequences which are located 3′ or downstream of the coding region and which are present on the mRNA are referred to as 3′ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene.
A “Sr50 gene” as used herein refers to a nucleotide sequence which is homologous to the isolated Sr50 gene or Sr50 cDNA (SEQ ID NO:9) described herein. As described herein, some alleles and variants of the Sr50 gene family encode a protein that confers resistance to stem rust (for example as caused by the Ug99 group of races of Puccinia graminis f. sp. tritici). Sr50 genes include the naturally occurring alleles or variants existing in cereals such as wheat. Nucleic acid molecules having the nucleotide sequence shown herein as SEQ ID NO:9 (cDNA) or SEQ ID NO:10 (open reading frame), encoding a protein with amino acid sequence SEQ ID NO:1, are examples of an Sr50 gene which confers resistance to stem rust.
A genomic form or clone of a gene containing the transcribed region may be interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences”, which may be either homologous or heterologous with respect to the “exons” of the gene. An “intron” as used herein is a segment of a gene which is transcribed as part of a primary RNA transcript but is not present in the mature mRNA molecule. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA). Introns may contain regulatory elements such as enhancers. “Exons” as used herein refer to the DNA regions corresponding to the RNA sequences which are present in the mature mRNA or the mature RNA molecule in cases where the RNA molecule is not translated. An mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term “gene” includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above. A gene may be introduced into an appropriate vector for extrachromosomal maintenance in a cell or, preferably, for integration into the host genome.
As used herein, a “chimeric gene” refers to any gene that comprises covalently joined sequences that are not found joined in nature. Typically, a chimeric gene comprises regulatory and transcribed or protein coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. The term “endogenous” is used herein to refer to a substance that is normally present or produced in an unmodified plant at the same developmental stage as the plant under investigation. An “endogenous gene” refers to a native gene in its natural location in the genome of an organism. As used herein, “recombinant nucleic acid molecule”, “recombinant polynucleotide” or variations thereof refer to a nucleic acid molecule which has been constructed or modified by recombinant DNA technology. The terms “foreign polynucleotide” or “exogenous polynucleotide” or “heterologous polynucleotide” and the like refer to any nucleic acid which is introduced into the genome of a cell by experimental manipulations.
Foreign or exogenous genes may be genes that are inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. The term “genetically modified” includes introducing genes into cells by transformation or transduction, mutating genes in cells and altering or modulating the regulation of a gene in a cell or organisms to which these acts have been done or their progeny.
Furthermore, the term “exogenous” in the context of a polynucleotide (nucleic acid) refers to the polynucleotide when present in a cell that does not naturally comprise the polynucleotide. The cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered amount of production of the encoded polypeptide, for example an exogenous polynucleotide which increases the expression of an endogenous polypeptide, or a cell which in its native state does not produce the polypeptide. Increased production of a polypeptide of the invention is also referred to herein as “over-expression”. An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components. The exogenous polynucleotide (nucleic acid) can be a contiguous stretch of nucleotides existing in nature, or comprise two or more contiguous stretches of nucleotides from different sources (naturally occurring and/or synthetic) joined to form a single polynucleotide. Typically such chimeric polynucleotides comprise at least an open reading frame encoding a polypeptide of the invention operably linked to a promoter suitable of driving transcription of the open reading frame in a cell of interest.
In an embodiment, the polynucleotide is not naturally occurring such as comprising nucleotides having a sequence as provided in SEQ ID NO:10. For example, in an embodiment the polynucleotide is a codon optimised polynucleotide encoding a polypeptide comprising amino acids having a sequence as provided in SEQ ID NO:1, a biologically active fragment thereof, or an amino acid sequence which is at least 82% identical, at least 90% identical, or at least 95% identical, to SEQ ID NO:1, for expression in a plant other than rye (such as wheat).
In an embodiment, if present in a rye plant, or part (such a ryegrain) or cell thereof, the polynucleotide is not present on chromosome 1RS.
The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 450 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 450 nucleotides. Preferably, the query sequence is at least 1,500 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 1,500 nucleotides. Even more preferably, the query sequence is at least 2,700 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 2,700 nucleotides. Even more preferably, the GAP analysis aligns two sequences over their entire length.
With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
In a further embodiment, the present invention relates to polynucleotides which are substantially identical to those specifically described herein. As used herein, with reference to a polynucleotide the term “substantially identical” means the substitution of one or a few (for example 2, 3, or 4) nucleotides whilst maintaining at least one activity of the native protein encoded by the polynucleotide. In addition, this term includes the addition or deletion of nucleotides which results in the increase or decrease in size of the encoded native protein by one or a few (for example 2, 3, or 4) amino acids whilst maintaining at least one activity of the native protein encoded by the polynucleotide.
The present invention also relates to the use of oligonucleotides, for instance in methods of screening for a polynucleotide of, or encoding a polypeptide of, the invention. As used herein, “oligonucleotides” are polynucleotides up to 50 nucleotides in length. The minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. They can be RNA, DNA, or combinations or derivatives of either. Oligonucleotides are typically relatively short single stranded molecules of 10 to 30 nucleotides, commonly 15-25 nucleotides in length. When used as a probe or as a primer in an amplification reaction, the minimum size of such an oligonucleotide is the size required for the formation of a stable hybrid between the oligonucleotide and a complementary sequence on a target nucleic acid molecule. Preferably, the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 25 nucleotides in length. Oligonucleotides of the present invention used as a probe are typically conjugated with a label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.
The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, or primers to produce nucleic acid molecules. Probes and/or primers can be used to clone homologues of the polynucleotides of the invention from other species. Furthermore, hybridization techniques known in the art can also be used to screen genomic or cDNA libraries for such homologues.
Polynucleotides and oligonucleotides of the present invention include those which hybridize under stringent conditions to one or more of the sequences provided as SEQ ID NO's: 9 and/or 10. As used herein, stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO4 at 50° C.; (2) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1% SDS.
Polynucleotides of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the nucleic acid). A variant of a polynucleotide or an oligonucleotide of the invention includes molecules of varying sizes of, and/or are capable of hybridising to, the wheat genome close to that of the reference polynucleotide or oligonucleotide molecules defined herein. For example, variants may comprise additional nucleotides (such as 1, 2, 3, 4, or more), or less nucleotides as long as they still hybridise to the target region. Furthermore, a few nucleotides may be substituted without influencing the ability of the oligonucleotide to hybridise to the target region. In addition, variants may readily be designed which hybridise close to, for example to within 50 nucleotides, the region of the plant genome where the specific oligonucleotides defined herein hybridise. In particular, this includes polynucleotides which encode the same polypeptide or amino acid sequence but which vary in nucleotide sequence by redundancy of the genetic code. The terms “polynucleotide variant” and “variant” also include naturally occurring allelic variants.
The present invention includes nucleic acid constructs comprising the polynucleotides of the invention, and vectors and host cells containing these, methods of their production and use, and uses thereof. The present invention refers to elements which are operably connected or linked. “Operably connected” or “operably linked” and the like refer to a linkage of polynucleotide elements in a functional relationship. Typically, operably connected nucleic acid sequences are contiguously linked and, where necessary to join two protein coding regions, contiguous and in reading frame. A coding sequence is “operably connected to” another coding sequence when RNA polymerase will transcribe the two coding sequences into a single RNA, which if translated is then translated into a single polypeptide having amino acids derived from both coding sequences. The coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein.
As used herein, the term “cis-acting sequence”, “cis-acting element” or “cis-regulatory region” or “regulatory region” or similar term shall be taken to mean any sequence of nucleotides, which when positioned appropriately and connected relative to an expressible genetic sequence, is capable of regulating, at least in part, the expression of the genetic sequence. Those skilled in the art will be aware that a cis-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of a gene sequence at the transcriptional or post-transcriptional level. In preferred embodiments of the present invention, the cis-acting sequence is an activator sequence that enhances or stimulates the expression of an expressible genetic sequence.
“Operably connecting” a promoter or enhancer element to a transcribable polynucleotide means placing the transcribable polynucleotide (e.g., protein-encoding polynucleotide or other transcript) under the regulatory control of a promoter, which then controls the transcription of that polynucleotide. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position a promoter or variant thereof at a distance from the transcription start site of the transcribable polynucleotide which is approximately the same as the distance between that promoter and the protein coding region 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 function. Similarly, the preferred positioning of a regulatory sequence element (e.g., an operator, enhancer etc) with respect to a transcribable polynucleotide 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.
“Promoter” or “promoter sequence” as used herein refers to a region of a gene, generally upstream (5′) of the RNA encoding region, which controls the initiation and level of transcription in the cell of interest. A “promoter” includes the transcriptional regulatory sequences of a classical genomic gene, such as a TATA box and CCAAT box sequences, as well as additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) that alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner A promoter is usually, but not necessarily (for example, some PolIII promoters), positioned upstream of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. Promoters may contain additional specific regulatory elements, located more distal to the start site to further enhance expression in a cell, and/or to alter the timing or inducibility of expression of a structural gene to which it is operably connected.
“Constitutive promoter” refers to a promoter that directs expression of an operably linked transcribed sequence in many or all tissues of an organism such as a plant. The term constitutive as used herein does not necessarily indicate that a gene is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, although some variation in level is often detectable. “Selective expression” as used herein refers to expression almost exclusively in specific organs of, for example, the plant, such as, for example, endosperm, embryo, leaves, fruit, tubers or root. In a preferred embodiment, a promoter is expressed selectively or preferentially in leaves and/or stems of a plant, preferably a cereal plant. Selective expression may therefore be contrasted with constitutive expression, which refers to expression in many or all tissues of a plant under most or all of the conditions experienced by the plant.
Selective expression may also result in compartmentation of the products of gene expression in specific plant tissues, organs or developmental stages. Compartmentation in specific subcellular locations such as the plastid, cytosol, vacuole, or apoplastic space may be achieved by the inclusion in the structure of the gene product of appropriate signals, eg. a signal peptide, for transport to the required cellular compartment, or in the case of the semi-autonomous organelles (plastids and mitochondria) by integration of the transgene with appropriate regulatory sequences directly into the organelle genome.
A “tissue-specific promoter” or “organ-specific promoter” is a promoter that is preferentially expressed in one tissue or organ relative to many other tissues or organs, preferably most if not all other tissues or organs in, for example, a plant. Typically, the promoter is expressed at a level 10-fold higher in the specific tissue or organ than in other tissues or organs.
In an embodiment, the promoter is a stem-specific promoter or a promoter which directs gene expression in an aerial part of the plant (green tissue specific promoter) such as a ribulose-1,5-bisphosphate carboxylase oxygenase (RUBISCO) promoter.
Examples of stem-specific promoters include, but are not limited to those described in U.S. Pat. No. 5,625,136, and Bam et al. (2008).
The promoters contemplated by the present invention may be native to the host plant to be transformed or may be derived from an alternative source, where the region is functional in the host plant. Other sources include the Agrobacterium T-DNA genes, such as the promoters of genes for the biosynthesis of nopaline, octapine, mannopine, or other opine promoters, tissue specific promoters (see, e.g., U.S. Pat. No. 5,459,252 and WO 91/13992); promoters from viruses (including host specific viruses), or partially or wholly synthetic promoters. Numerous promoters that are functional in mono- and dicotyledonous plants are well known in the art (see, for example, Greve, 1983; Salomon et al., 1984; Garfinkel et al., 1983; Barker et al., 1983); including various promoters isolated from plants and viruses such as the cauliflower mosaic virus promoter (CaMV 35S, 19S). Non-limiting methods for assessing promoter activity are disclosed by Medberry et al. (1992, 1993), Sambrook et al. (1989, supra) and U.S. Pat. No. 5,164,316.
Alternatively or additionally, the promoter may be an inducible promoter or a developmentally regulated promoter which is capable of driving expression of the introduced polynucleotide at an appropriate developmental stage of the, for example, plant. Other cis-acting sequences which may be employed include transcriptional and/or translational enhancers. Enhancer regions are well known to persons skilled in the art, and can include an ATG translational initiation codon and adjacent sequences. When included, the initiation codon should be in phase with the reading frame of the coding sequence relating to the foreign or exogenous polynucleotide to ensure translation of the entire sequence if it is to be translated. Translational initiation regions may be provided from the source of the transcriptional initiation region, or from a foreign or exogenous polynucleotide. The sequence can also be derived from the source of the promoter selected to drive transcription, and can be specifically modified so as to increase translation of the mRNA.
The nucleic acid construct of the present invention may comprise a 3′ non-translated sequence from about 50 to 1,000 nucleotide base pairs which may include a transcription termination sequence. A 3′ non-translated sequence may contain a transcription termination signal which may or may not include a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing. A polyadenylation signal functions for addition of polyadenylic acid tracts to the 3′ end of a mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5′ AATAAA-3′ although variations are not uncommon. Transcription termination sequences which do not include a polyadenylation signal include terminators for Poll or PolIII RNA polymerase which comprise a run of four or more thymidines. Examples of suitable 3′ non-translated sequences are the 3′ transcribed non-translated regions containing a polyadenylation signal from an octopine synthase (ocs) gene or nopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan et al., 1983). Suitable 3′ non-translated sequences may also be derived from plant genes such as the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene, although other 3′ elements known to those of skill in the art can also be employed.
As the DNA sequence inserted between the transcription initiation site and the start of the coding sequence, i.e., the untranslated 5′ leader sequence (5′UTR), can influence gene expression if it is translated as well as transcribed, one can also employ a particular leader sequence. Suitable leader sequences include those that comprise sequences selected to direct optimum expression of the foreign or endogenous DNA sequence. For example, such leader sequences include a preferred consensus sequence which can increase or maintain mRNA stability and prevent inappropriate initiation of translation as for example described by Joshi (1987).
The present invention includes use of vectors for manipulation or transfer of genetic constructs. By “chimeric vector” is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably is double-stranded DNA and contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or capable of integration into the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into a cell, is integrated into the genome of the recipient cell and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene, a herbicide resistance gene or other gene that can be used for selection of suitable transformants Examples of such genes are well known to those of skill in the art.
The nucleic acid construct of the invention can be introduced into a vector, such as a plasmid. Plasmid vectors typically include additional nucleic acid sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, or binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of prokaryotic and eukaryotic (especially plant) cells.
By “marker gene” is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker. A selectable marker gene confers a trait for which one can “select” based on resistance to a selective agent (e.g., a herbicide, antibiotic, radiation, heat, or other treatment damaging to untransformed cells). A screenable marker gene (or reporter gene) confers a trait that one can identify through observation or testing, i.e., by “screening” (e.g., β-glucuronidase, luciferase, GFP or other enzyme activity not present in untransformed cells). The marker gene and the nucleotide sequence of interest do not have to be linked.
To facilitate identification of transformants, the nucleic acid construct desirably comprises a selectable or screenable marker gene as, or in addition to, the foreign or exogenous polynucleotide. The actual choice of a marker is not crucial as long as it is functional (i.e., selective) in combination with the plant cells of choice. The marker gene and the foreign or exogenous polynucleotide of interest do not have to be linked, since co-transformation of unlinked genes as, for example, described in U.S. Pat. No. 4,399,216 is also an efficient process in plant transformation.
Examples of bacterial selectable markers are markers that confer antibiotic resistance such as ampicillin, erythromycin, chloramphenicol or tetracycline resistance, preferably kanamycin resistance. Exemplary selectable markers for selection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin phosphotransferase (nptII) gene conferring resistance to kanamycin, paromomycin, G418; a glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides as, for example, described in EP 256223; a glutamine synthetase gene conferring, upon overexpression, resistance to glutamine synthetase inhibitors such as phosphinothricin as, for example, described in WO 87/05327, an acetyltransferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin as, for example, described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as, for example, described by Hinchee et al. (1988), a bar gene conferring resistance against bialaphos as, for example, described in WO91/02071; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al., 1988); a mutant acetolactate synthase gene (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP 154,204); a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide.
Preferred screenable markers include, but are not limited to, a uidA gene encoding a β-glucuronidase (GUS) enzyme for which various chromogenic substrates are known, a β-galactosidase gene encoding an enzyme for which chromogenic substrates are known, an aequorin gene (Prasher et al., 1985), which may be employed in calcium-sensitive bioluminescence detection; a green fluorescent protein gene (Niedz et al., 1995) or derivatives thereof; a luciferase (luc) gene (Ow et al., 1986), which allows for bioluminescence detection, and others known in the art. By “reporter molecule” as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that facilitates determination of promoter activity by reference to protein product.
Preferably, the nucleic acid construct is stably incorporated into the genome of, for example, the plant. Accordingly, the nucleic acid comprises appropriate elements which allow the molecule to be incorporated into the genome, or the construct is placed in an appropriate vector which can be incorporated into a chromosome of a plant cell.
One embodiment of the present invention includes a recombinant vector, which includes at least one polynucleotide molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
The level of a protein of the invention may be modulated by increasing the level of expression of a nucleotide sequence that codes for the protein in a plant cell, or decreasing the level of expression of a gene encoding the protein in the plant, leading to modified pathogen resistance. The level of expression of a gene may be modulated by altering the copy number per cell, for example by introducing a synthetic genetic construct comprising the coding sequence and a transcriptional control element that is operably connected thereto and that is functional in the cell. A plurality of transformants may be selected and screened for those with a favourable level and/or specificity of transgene expression arising from influences of endogenous sequences in the vicinity of the transgene integration site. A favourable level and pattern of transgene expression is one which results in a substantial modification of pathogen resistance or other phenotype. Alternatively, a population of mutagenized seed or a population of plants from a breeding program may be screened for individual lines with altered pathogen resistance or other phenotype associated with pathogen resistance.
Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention, or progeny cells thereof. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Preferred host cells are plant cells, more preferably cells of a cereal plant, more preferably barley or wheat cells, and even more preferably a wheat cell.
The term “plant” as used herein as a noun refers to whole plants and refers to any member of the Kingdom Plantae, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like. Plantlets and germinated seeds from which roots and shoots have emerged are also included within the meaning of “plant”. The term “plant parts” as used herein refers to one or more plant tissues or organs which are obtained from a plant and which comprises genomic DNA of the plant. Plant parts include vegetative structures (for example, leaves, stems), roots, floral organs/structures, seed (including embryo, cotyledons, and seed coat), plant tissue (for example, vascular tissue, ground tissue, and the like), cells and progeny of the same. The term “plant cell” as used herein refers to a cell obtained from a plant or in a plant and includes protoplasts or other cells derived from plants, gamete-producing cells, and cells which regenerate into whole plants. Plant cells may be cells in culture. By “plant tissue” is meant differentiated tissue in a plant or obtained from a plant (“explant”) or undifferentiated tissue derived from immature or mature embryos, seeds, roots, shoots, fruits, tubers, pollen, tumor tissue, such as crown galls, and various forms of aggregations of plant cells in culture, such as calli. Exemplary plant tissues in or from seeds are cotyledon, embryo and embryo axis. The invention accordingly includes plants and plant parts and products comprising these.
As used herein, the term “seed” refers to “mature seed” of a plant, which is either ready for harvesting or has been harvested from the plant, such as is typically harvested commercially in the field, or as “developing seed” which occurs in a plant after fertilisation and prior to seed dormancy being established and before harvest.
A “transgenic plant” as used herein refers to a plant that contains a nucleic acid construct not found in a wild-type plant of the same species, variety or cultivar. That is, transgenic plants (transformed plants) contain genetic material (a transgene) that they did not contain prior to the transformation. The transgene may include genetic sequences obtained from or derived from a plant cell, or another plant cell, or a non-plant source, or a synthetic sequence. Typically, the transgene has been introduced into the plant by human manipulation such as, for example, by transformation but any method can be used as one of skill in the art recognizes. The genetic material is preferably stably integrated into the genome of the plant. The introduced genetic material may comprise sequences that naturally occur in the same species but in a rearranged order or in a different arrangement of elements, for example an antisense sequence. Plants containing such sequences are included herein in “transgenic plants”.
A “non-transgenic plant” is one which has not been genetically modified by the introduction of genetic material by recombinant DNA techniques. In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired phenotype.
As used herein, the term “compared to an isogenic plant”, or similar phrases, refers to a plant which is isogenic relative to the transgenic plant but without the transgene of interest. Preferably, the corresponding non-transgenic plant is of the same cultivar or variety as the progenitor of the transgenic plant of interest, or a sibling plant line which lacks the construct, often termed a “segregant”, or a plant of the same cultivar or variety transformed with an “empty vector” construct, and may be a non-transgenic plant. “Wild type”, as used herein, refers to a cell, tissue or plant that has not been modified according to the invention. Wild-type cells, tissue or plants may be used as controls to compare levels of expression of an exogenous nucleic acid or the extent and nature of trait modification with cells, tissue or plants modified as described herein.
Transgenic plants, as defined in the context of the present invention include progeny of the plants which have been genetically modified using recombinant techniques, wherein the progeny comprise the transgene of interest. Such progeny may be obtained by self-fertilisation of the primary transgenic plant or by crossing such plants with another plant of the same species. This would generally be to modulate the production of at least one protein defined herein in the desired plant or plant organ. Transgenic plant parts include all parts and cells of said plants comprising the transgene such as, for example, cultured tissues, callus and protoplasts.
Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. Target plants include, but are not limited to, the following: cereals (for example, wheat, barley, rye, oats, rice, maize, sorghum and related crops); beet (sugar beet and fodder beet); pomes, stone fruit and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and black-berries); leguminous plants (beans, lentils, peas, soybeans); oil plants (rape or other Brassicas, mustard, poppy, olives, sunflowers, safflower, flax, coconut, castor oil plants, cocoa beans, groundnuts); cucumber plants (marrows, cucumbers, melons); fibre plants (cotton, flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit, mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, paprika); lauraceae (avocados, cinnamon, camphor); or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, turf, bananas and natural rubber plants, as well as ornamentals (flowers, shrubs, broad-leaved trees and evergreens, such as conifers). Preferably, the plant is a cereal plant, more preferably wheat, rice, maize, triticale, oats, sorghum or barley, even more preferably wheat.
As used herein, the term “wheat” refers to any species of the Genus Triticum, including progenitors thereof, as well as progeny thereof produced by crosses with other species. Wheat includes “hexaploid wheat” which has genome organization of AABBDD, comprised of 42 chromosomes, and “tetraploid wheat” which has genome organization of AABB, comprised of 28 chromosomes. Hexaploid wheat includes T. aestivum, T. spelta, T. macha, T. compactum, T. sphaerococcum, T. vavilovii, and interspecies cross thereof. A preferred species of hexaploid wheat is T. aestivum ssp aestivum (also termed “breadwheat”). Tetraploid wheat includes T. durum (also referred to herein as durum wheat or Triticum turgidum ssp. durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspecies cross thereof. In addition, the term “wheat” includes potential progenitors of hexaploid or tetraploid Triticum sp. such as T. uartu, T. monococcum or T. boeoticum for the A genome, Aegilops speltoides for the B genome, and T. tauschii (also known as Aegilops squarrosa or Aegilops tauschii) for the D genome. Particularly preferred progenitors are those of the A genome, even more preferably the A genome progenitor is T. monococcum. A wheat cultivar for use in the present invention may belong to, but is not limited to, any of the above-listed species. Also encompassed are plants that are produced by conventional techniques using Triticum sp. as a parent in a sexual cross with a non-Triticum species (such as rye [Secale cereale]), including but not limited to Triticale.
As used herein, the term “barley” refers to any species of the Genus Hordeum, including progenitors thereof, as well as progeny thereof produced by crosses with other species. It is preferred that the plant is of a Hordeum species which is commercially cultivated such as, for example, a strain or cultivar or variety of Hordeum vulgare or suitable for commercial production of grain.
Transgenic plants, as defined in the context of the present invention include plants (as well as parts and cells of said plants) and their progeny which have been genetically modified using recombinant techniques to cause production of at least one polypeptide of the present invention in the desired plant or plant organ. Transgenic plants can be produced using techniques known in the art, such as those generally described in A. Slater et al., Plant Biotechnology—The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).
In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired phenotype. The transgenic plants may also be heterozygous for the introduced transgene(s), such as, for example, in F1 progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.
As used herein, the “other genetic markers” may be any molecules which are linked to a desired trait of a plant. Such markers are well known to those skilled in the art and include molecular markers linked to genes determining traits such disease resistance, yield, plant morphology, grain quality, dormancy traits, grain colour, gibberellic acid content in the seed, plant height, flour colour and the like. Examples of such genes are the stripe rust resistance genes Yr10 or Yr17, the nematode resistance genes such as Cre1 and Cre3, alleles at glutenin loci that determine dough strength such as Ax, Bx, Dx, Ay, By and Dy alleles, the Rht genes that determine a semi-dwarf growth habit and therefore lodging resistance.
Four general methods for direct delivery of a gene into cells have been described: (1) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980); electroporation (see, for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO 92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat. Nos. 4,945,050 and 5,141,131); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms (Curiel et al., 1992; Wagner et al., 1992).
Acceleration methods that may be used include, for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required. A particle delivery system suitable for use with the present invention is the helium acceleration PDS-1000/He gun is available from Bio-Rad Laboratories. For the bombardment, immature embryos or derived target cells such as scutella or calli from immature embryos may be arranged on solid culture medium.
In another alternative embodiment, plastids can be stably transformed. Method disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (U.S. Pat. Nos. 5,451,513, 5,545,818, 5,877,402, 5,932,479, and WO 99/05265.
Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, U.S. Pat. Nos. 5,177,010, 5,104,310, 5,004,863, 5,159,135). Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome.
Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., Plant DNA Infectious Agents, Hohn and Schell, (editors), Springer-Verlag, New York, (1985): 179-203). Moreover, technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant varieties where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
A transgenic plant formed using Agrobacterium transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.
It is also to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both exogenous genes. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, Breeding Methods for Cultivar Development, J. Wilcox (editor) American Society of Agronomy, Madison Wis. (1987).
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. Application of these systems to different plant varieties depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).
Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.
The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., Methods for Plant Molecular Biology, Academic Press, San Diego, (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired exogenous nucleic acid is cultivated using methods well known to one skilled in the art.
Methods for transforming dicots, primarily by use of Agrobacterium tumefaciens, and obtaining transgenic plants have been published for cotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908); soybean (U.S. Pat. Nos. 5,569,834, 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., 1996); and pea (Grant et al., 1995).
Methods for transformation of cereal plants such as wheat and barley for introducing genetic variation into the plant by introduction of an exogenous nucleic acid and for regeneration of plants from protoplasts or immature plant embryos are well known in the art, see for example, CA 2,092,588, AU 61781/94, AU 667939, U.S. Pat. No. 6,100,447, WO 97/048814, U.S. Pat. Nos. 5,589,617, 6,541,257, and other methods are set out in WO 99/14314. Preferably, transgenic wheat or barley plants are produced by Agrobacterium tumefaciens mediated transformation procedures. Vectors carrying the desired nucleic acid construct may be introduced into regenerable wheat cells of tissue cultured plants or explants, or suitable plant systems such as protoplasts. The regenerable wheat cells are preferably from the scutellum of immature embryos, mature embryos, callus derived from these, or the meristematic tissue.
To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way to quantitate protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.
Marker assisted selection is a well recognised method of selecting for heterozygous plants required when backcrossing with a recurrent parent in a classical breeding program. The population of plants in each backcross generation will be heterozygous for the gene of interest normally present in a 1:1 ratio in a backcross population, and the molecular marker can be used to distinguish the two alleles of the gene. By extracting DNA from, for example, young shoots and testing with a specific marker for the introgressed desirable trait, early selection of plants for further backcrossing is made whilst energy and resources are concentrated on fewer plants. To further speed up the backcrossing program, the embryo from immature seeds (25 days post anthesis) may be excised and grown up on nutrient media under sterile conditions, rather than allowing full seed maturity. This process, termed “embryo rescue”, used in combination with DNA extraction at the three leaf stage and analysis of at least one Sr50 allele or variant that confers enhanced resistance to stem rust to the plant, allows rapid selection of plants carrying the desired trait, which may be nurtured to maturity in the greenhouse or field for subsequent further backcrossing to the recurrent parent.
Any molecular biological technique known in the art can be used in the methods of the present invention. Such methods include, but are not limited to, the use of nucleic acid amplification, nucleic acid sequencing, nucleic acid hybridization with suitably labeled probes, single-strand conformational analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalytic nucleic acid cleavage or a combination thereof (see, for example, Lemieux, 2000; Langridge et al., 2001). The invention also includes the use of molecular marker techniques to detect polymorphisms linked to alleles of the (for example) Sr50 gene which confers enhanced resistance to stem rust. Such methods include the detection or analysis of restriction fragment length polymorphisms (RFLP), RAPD, amplified fragment length polymorphisms (AFLP) and microsatellite (simple sequence repeat, SSR) polymorphisms. The closely linked markers can be obtained readily by methods well known in the art, such as Bulked Segregant Analysis, as reviewed by Langridge et al. (2001).
In an embodiment, a linked loci for marker assisted selection is at least within 1 cM, or 0.5 cM, or 0.1 cM, or 0.01 cM from a gene encoding a polypeptide of the invention.
The “polymerase chain reaction” (“PCR”) is a reaction in which replicate copies are made of a target polynucleotide using a “pair of primers” or “set of primers” consisting of “upstream” and a “downstream” primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme. Methods for PCR are known in the art, and are taught, for example, in “PCR” (M. J. McPherson and S. G Moller (editors), BIOS Scientific Publishers Ltd, Oxford, (2000)). PCR can be performed on cDNA obtained from reverse transcribing mRNA isolated from plant cells expressing a Sr50 gene or allele which confers enhanced resistance to stem rust. However, it will generally be easier if PCR is performed on genomic DNA isolated from a plant.
A primer is an oligonucleotide sequence that is capable of hybridising in a sequence specific fashion to the target sequence and being extended during the PCR. Amplicons or PCR products or PCR fragments or amplification products are extension products that comprise the primer and the newly synthesized copies of the target sequences. Multiplex PCR systems contain multiple sets of primers that result in simultaneous production of more than one amplicon. Primers may be perfectly matched to the target sequence or they may contain internal mismatched bases that can result in the introduction of restriction enzyme or catalytic nucleic acid recognition/cleavage sites in specific target sequences. Primers may also contain additional sequences and/or contain modified or labelled nucleotides to facilitate capture or detection of amplicons. Repeated cycles of heat denaturation of the DNA, annealing of primers to their complementary sequences and extension of the annealed primers with polymerase result in exponential amplification of the target sequence. The terms target or target sequence or template refer to nucleic acid sequences which are amplified.
Methods for direct sequencing of nucleotide sequences are well known to those skilled in the art and can be found for example in Ausubel et al., (supra) and Sambrook et al., (supra). Sequencing can be carried out by any suitable method, for example, dideoxy sequencing, chemical sequencing or variations thereof. Direct sequencing has the advantage of determining variation in any base pair of a particular sequence.
Plants of the invention can be produced using the process known as TILLING (Targeting Induced Local Lesions IN Genomes). In a first step, introduced mutations such as novel single base pair changes are induced in a population of plants by treating seeds (or pollen) with a chemical mutagen, and then advancing plants to a generation where mutations will be stably inherited. DNA is extracted, and seeds are stored from all members of the population to create a resource that can be accessed repeatedly over time.
For a TILLING assay, PCR primers are designed to specifically amplify a single gene target of interest. Specificity is especially important if a target is a member of a gene family or part of a polyploid genome. Next, dye-labeled primers can be used to amplify PCR products from pooled DNA of multiple individuals. These PCR products are denatured and reannealed to allow the formation of mismatched base pairs. Mismatches, or heteroduplexes, represent both naturally occurring single nucleotide polymorphisms (SNPs) (i.e., several plants from the population are likely to carry the same polymorphism) and induced SNPs (i.e., only rare individual plants are likely to display the mutation). After heteroduplex formation, the use of an endonuclease, such as Cel I, that recognizes and cleaves mismatched DNA is the key to discovering novel SNPs within a TILLING population.
Using this approach, many thousands of plants can be screened to identify any individual with a single base change as well as small insertions or deletions (1-30 bp) in any gene or specific region of the genome. Genomic fragments being assayed can range in size anywhere from 0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the ends of fragments where SNP detection is problematic due to noise) and 96 lanes per assay, this combination allows up to a million base pairs of genomic DNA to be screened per single assay, making TILLING a high-throughput technique.
TILLING is further described in Slade and Knauf (2005), and Henikoff et al. (2004). In addition to allowing efficient detection of mutations, high-throughput TILLING technology is ideal for the detection of natural polymorphisms. Therefore, interrogating an unknown homologous DNA by heteroduplexing to a known sequence reveals the number and position of polymorphic sites. Both nucleotide changes and small insertions and deletions are identified, including at least some repeat number polymorphisms. This has been called Ecotilling (Comai et al., 2004).
Each SNP is recorded by its approximate position within a few nucleotides. Thus, each haplotype can be archived based on its mobility. Sequence data can be obtained with a relatively small incremental effort using aliquots of the same amplified DNA that is used for the mismatch-cleavage assay. The left or right sequencing primer for a single reaction is chosen by its proximity to the polymorphism. Sequencher software performs a multiple alignment and discovers the base change, which in each case confirmed the gel band.
Ecotilling can be performed more cheaply than full sequencing, the method currently used for most SNP discovery. Plates containing arrayed ecotypic DNA can be screened rather than pools of DNA from mutagenized plants. Because detection is on gels with nearly base pair resolution and background patterns are uniform across lanes, bands that are of identical size can be matched, thus discovering and genotyping SNPs in a single step. In this way, ultimate sequencing of the SNP is simple and efficient, made more so by the fact that the aliquots of the same PCR products used for screening can be subjected to DNA sequencing.
Grain/seed of the invention, preferably cereal grain and more preferably wheat grain, or other plant parts of the invention, can be processed to produce a food ingredient, food or non-food product using any technique known in the art.
In one embodiment, the product is whole grain flour such as, for example, an ultrafine-milled whole grain flour, or a flour made from about 100% of the grain. The whole grain flour includes a refined flour constituent (refined flour or refined flour) and a coarse fraction (an ultrafine-milled coarse fraction).
Refined flour may be flour which is prepared, for example, by grinding and bolting cleaned grain such as wheat or barley grain. The particle size of refined flour is described as flour in which not less than 98% passes through a cloth having openings not larger than those of woven wire cloth designated “212 micrometers (U.S. Wire 70)”. The coarse fraction includes at least one of: bran and germ. For instance, the germ is an embryonic plant found within the grain kernel. The germ includes lipids, fiber, vitamins, protein, minerals and phytonutrients, such as flavonoids. The bran includes several cell layers and has a significant amount of lipids, fiber, vitamins, protein, minerals and phytonutrients, such as flavonoids. Further, the coarse fraction may include an aleurone layer which also includes lipids, fiber, vitamins, protein, minerals and phytonutrients, such as flavonoids. The aleurone layer, while technically considered part of the endosperm, exhibits many of the same characteristics as the bran and therefore is typically removed with the bran and germ during the milling process. The aleurone layer contains proteins, vitamins and phytonutrients, such as ferulic acid.
Further, the coarse fraction may be blended with the refined flour constituent. The coarse fraction may be mixed with the refined flour constituent to form the whole grain flour, thus providing a whole grain flour with increased nutritional value, fiber content, and antioxidant capacity as compared to refined flour. For example, the coarse fraction or whole grain flour may be used in various amounts to replace refined or whole grain flour in baked goods, snack products, and food products. The whole grain flour of the present invention (i.e.—ultrafine-milled whole grain flour) may also be marketed directly to consumers for use in their homemade baked products. In an exemplary embodiment, a granulation profile of the whole grain flour is such that 98% of particles by weight of the whole grain flour are less than 212 micrometers.
In further embodiments, enzymes found within the bran and germ of the whole grain flour and/or coarse fraction are inactivated in order to stabilize the whole grain flour and/or coarse fraction. Stabilization is a process that uses steam, heat, radiation, or other treatments to inactivate the enzymes found in the bran and germ layer. Flour that has been stabilized retains its cooking characteristics and has a longer shelf life.
In additional embodiments, the whole grain flour, the coarse fraction, or the refined flour may be a component (ingredient) of a food product and may be used to product a food product. For example, the food product may be a bagel, a biscuit, a bread, a bun, a croissant, a dumpling, an English muffin, a muffin, a pita bread, a quickbread, a refrigerated/frozen dough product, dough, baked beans, a burrito, chili, a taco, a tamale, a tortilla, a pot pie, a ready to eat cereal, a ready to eat meal, stuffing, a microwaveable meal, a brownie, a cake, a cheesecake, a coffee cake, a cookie, a dessert, a pastry, a sweet roll, a candy bar, a pie crust, pie filling, baby food, a baking mix, a batter, a breading, a gravy mix, a meat extender, a meat substitute, a seasoning mix, a soup mix, a gravy, a roux, a salad dressing, a soup, sour cream, a noodle, a pasta, ramen noodles, chow mein noodles, lo mein noodles, an ice cream inclusion, an ice cream bar, an ice cream cone, an ice cream sandwich, a cracker, a crouton, a doughnut, an egg roll, an extruded snack, a fruit and grain bar, a microwaveable snack product, a nutritional bar, a pancake, a par-baked bakery product, a pretzel, a pudding, a granola-based product, a snack chip, a snack food, a snack mix, a waffle, a pizza crust, animal food or pet food.
In alternative embodiments, the whole grain flour, refined flour, or coarse fraction may be a component of a nutritional supplement. For instance, the nutritional supplement may be a product that is added to the diet containing one or more additional ingredients, typically including: vitamins, minerals, herbs, amino acids, enzymes, antioxidants, herbs, spices, probiotics, extracts, prebiotics and fiber. The whole grain flour, refined flour or coarse fraction of the present invention includes vitamins, minerals, amino acids, enzymes, and fiber. For instance, the coarse fraction contains a concentrated amount of dietary fiber as well as other essential nutrients, such as B-vitamins, selenium, chromium, manganese, magnesium, and antioxidants, which are essential for a healthy diet. For example 22 grams of the coarse fraction of the present invention delivers 33% of an individual's daily recommend consumption of fiber. The nutritional supplement may include any known nutritional ingredients that will aid in the overall health of an individual, examples include but are not limited to vitamins, minerals, other fiber components, fatty acids, antioxidants, amino acids, peptides, proteins, lutein, ribose, omega-3 fatty acids, and/or other nutritional ingredients. The supplement may be delivered in, but is not limited to the following forms: instant beverage mixes, ready-to-drink beverages, nutritional bars, wafers, cookies, crackers, gel shots, capsules, chews, chewable tablets, and pills. One embodiment delivers the fiber supplement in the form of a flavored shake or malt type beverage, this embodiment may be particularly attractive as a fiber supplement for children.
In an additional embodiment, a milling process may be used to make a multi-grain flour or a multi-grain coarse fraction. For example, bran and germ from one type of grain may be ground and blended with ground endosperm or whole grain cereal flour of another type of cereal. Alternatively bran and germ of one type of grain may be ground and blended with ground endosperm or whole grain flour of another type of grain. It is contemplated that the present invention encompasses mixing any combination of one or more of bran, germ, endosperm, and whole grain flour of one or more grains. This multi-grain approach may be used to make custom flour and capitalize on the qualities and nutritional contents of multiple types of cereal grains to make one flour.
It is contemplated that the whole grain flour, coarse fraction and/or grain products of the present invention may be produced by any milling process known in the art. An exemplary embodiment involves grinding grain in a single stream without separating endosperm, bran, and germ of the grain into separate streams. Clean and tempered grain is conveyed to a first passage grinder, such as a hammermill, roller mill, pin mill, impact mill, disc mill, air attrition mill, gap mill, or the like. After grinding, the grain is discharged and conveyed to a sifter. Further, it is contemplated that the whole grain flour, coarse fraction and/or grain products of the present invention may be modified or enhanced by way of numerous other processes such as: fermentation, instantizing, extrusion, encapsulation, toasting, roasting, or the like.
A malt-based beverage provided by the present invention involves alcohol beverages (including distilled beverages) and non-alcohol beverages that are produced by using malt as a part or whole of their starting material. Examples include beer, happoshu (low-malt beer beverage), whisky, low-alcohol malt-based beverages (e.g., malt-based beverages containing less than 1% of alcohols), and non-alcohol beverages.
Malting is a process of controlled steeping and germination followed by drying of the grain such as barley and wheat grain. This sequence of events is important for the synthesis of numerous enzymes that cause grain modification, a process that principally depolymerizes the dead endosperm cell walls and mobilizes the grain nutrients. In the subsequent drying process, flavour and colour are produced due to chemical browning reactions. Although the primary use of malt is for beverage production, it can also be utilized in other industrial processes, for example as an enzyme source in the baking industry, or as a flavouring and colouring agent in the food industry, for example as malt or as a malt flour, or indirectly as a malt syrup, etc.
In one embodiment, the present invention relates to methods of producing a malt composition. The method preferably comprises the steps of:
(i) providing grain, such as barley or wheat grain, of the invention,
(ii) steeping said grain,
(iii) germinating the steeped grains under predetermined conditions and
(iv) drying said germinated grains.
For example, the malt may be produced by any of the methods described in Hoseney (Principles of Cereal Science and Technology, Second Edition, 1994: American Association of Cereal Chemists, St. Paul, Minn.) However, any other suitable method for producing malt may also be used with the present invention, such as methods for production of speciality malts, including, but limited to, methods of roasting the malt.
Malt is mainly used for brewing beer, but also for the production of distilled spirits. Brewing comprises wort production, main and secondary fermentations and post-treatment. First the malt is milled, stirred into water and heated. During this “mashing”, the enzymes activated in the malting degrade the starch of the kernel into fermentable sugars. The produced wort is clarified, yeast is added, the mixture is fermented and a post-treatment is performed.
Plants of two wheat lines containing independent Sr50 introgressions, Gabo1BL.1RS and Gabo1DL.1RS-DR.A1, and of the mutants M2 (00.002), M7 (00.007) and M13 (01.013) derived from these lines by y-irradiation and ethyl methanesulfonate (EMS) treatment were used in the experiments described herein. These lines were as described by Rogowosky et al. (1991) and Mago et al. (2004). The Gabo1BL.1RS translocation was backcrossed five generations into wheat plants of the cultivar Federation to generate the resistance line Federation*5/Gabo1BL.1RS-1-1 containing Sr50 in the absence of the Gabo background genes. Plants of the Gabo1BL.1RS line were resistant to North American and African Pgt rust isolates by the presence of the Sr50 resistance gene.
Nicotiana benthamiana plants were grown in a growth chamber at 22° C. with a 16 hours light period, 8 hours dark per 24 hours.
Markers developed from an Mla gene-containing BAC from chromosome 5H of barley (Wei et al., 1999) and used for mutant analysis herein were as described by Mago et al. (2004). Stem rust phenotyping and mutant screening were done on 1 week old seedlings with Puccinia graminis f. sp tritici (Pgt) race 98-1 2,3,5,6 (Sydney University culture accession 279) as described in Mago et al. (2009). To differentiate between Sr50 and Sr31, plants were phenotyped with Pgt races TTKSK (Ug99), TTKST and 98-1,2,3,5,7+50 (Sydney University culture accession 632). Transgenic plants were also phenotyped for leaf rust and stripe rust responses by inoculating seedlings with P. triticina pathotype 104-2,3,(6),(7),11 (Sydney University culture accession 423) and P. striiformis f. sp. tritici pathotype 110 E143A+(Sydney University culture accession 444), respectively.
For rust infection assays to distinguish Sr50 from other resistance genes with different specificities, one-week-old seedlings were inoculated with Pgt races 34-2,4,5,7,11, (Plant Breeding Institute accession #760785); 34-2,12,13, (#840552); 126-5,6,7,11, (#334), TTKSK (04KEN156/04, Ug99), TTKST (06KEN19v3, Ug99+Sr24), QFCSC (03ND76C), TPMKC (74MN1409), TRTTF (06YEM34-1), TKKTP (13GER16-1), QCMJC (07WA140-515). Plants were also inoculated with P. triticina race 104-2,3,(6),(7),11 (#890172) and P. striiformis f. sp. tritici race 110 E143 A+(#861725). Infection types were scored as described (McIntosh, 1995).
DraI-digested genomic DNA preparations from wheat plants of the line Gabo1DL.1RS were electrophoresed on a 1% agarose gel overnight and the region of the gel containing fragments of between 9-13 kb was excised and DNA extracted and purified from the gel. The resulting DNA was ligated to BamHI adaptors and cloned into an EMBL3 X-BamHI vector (Epicentre Technologies) and packaged using the MaxPlax (Epicentre Technologies) lambda packaging extracts according to the manufacturer's instructions. The library was hybridised with a probe derived from the LRR-encoding region of Mla1 (B76: Mago et al., 2004) and positive clones were identified and sequenced.
A wheat-rye ditelosomic addition line comprising a rye chromosome 1RS carrying Sr50 in the background of wheat cv. Chinese Spring was described by Simkova et al. (2008). A BAC library was prepared from flow sorted chromosome 1RS from this ditelosomic addition line. This 1RS chromosome-specific library was screened by DNA hybridisation using the B76 probe as described below. Positive BAC clones were purified and fingerprinted using high-information content BAC fingerprinting according to standard methods. BAC DNA was prepared using a modified alkaline lysis protocol (Sinnett et al., 1998). BAC end sequencing of clones in the minimal tiling path of the contig containing Sr50 was performed using primers designed to the pindigo BAC vector using Sanger sequencing. BAC sequences were used to design specific PCR primers. Five BAC clones were sequenced using the Roche 454 sequencing platform. Repeat sequences present in the assembled BACs were masked using the Wheat Repeats Database (wheat.pw.usda.gov/ITMI/Repeats/blastrepeats3.html). Sequence reads were assembled using Newbler v2.3. Non-repeated sequences were analysed for genes using the gene prediction softwares FGENESH (www.softberry.com) and GENSCAN (genes.mit.edu/GENSCAN.html).
PCR amplification of candidate rust resistance genes from Gabo 1DL.1RS-DR.A1 and various susceptible M2 mutants used primer pairs flanking the genes. Amplified sequences were compared for nucleotide variations using multiple sequence alignment (CLUSTAL-European Bioinformatics Institute—www.edi.ac.uk/Tools/sequence.html). RNA extraction, cDNA synthesis, 5′ and 3′ RACE (rapid amplification of cDNA ends) were done using the methods described in Periyannan et al. (2013). Primers designed at the predicted 5′ and 3′ termini of Sr50 transcripts were used for RT-PCR analysis. For 5′- and 3′-RACE, primers designed at the 5′ and 3′ coding regions were used as the gene-specific primers.
Two genomic DNA constructs each comprising Sr50 and therefore encoding the Sr50 polypeptide were generated. The first contained a 7.5 kb fragment including 2.4 kbp upstream and 1.38 kbp downstream regions relative to the protein coding region of ScRGA1-A in the binary vector pVecBarll. This fragment was amplified from Gabo1DL.1RS genomic DNA using primers F1-R1 followed by nested primers F2-R2 listed in Table 2 with PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies) under the manufacturer's recommended conditions. The second construct contained a 9.8 kbp NotI fragment from BAC clone 180C18 including 4.2 kbp upstream and 1.88kbp downstream regions relative to the protein coding region and inserted into the binary vector pVecNeo.
Binary vectors pVecNeo and pVecBarll are derivatives of pWBvec8 (Wang et al., 1998) in which the 35S promoter::hygromycin resistance gene was replaced with a 35S promoter::NPTII selectable marker gene derived from pCMneoSTL2 (Maas et al., 1997), or the bialaphos resistance gene (bar) coding region as a selection marker for plant transformation.
Transformation of the stem rust susceptible wheat cultivar Fielder was done using Agrobacterium tumefaciens strain GV3101 (pMP90) as described (Ishida et al., 2014; Richardson et al., 2014). TO transformants and T1 progeny plants, including both plants which comprised the transgene and segregants that lacked it as negative control plants, were tested for rust response with Pgt strain 98-1,2,3,5,6 as described above. The presence of the transgene and/or selectable marker gene was detected by Southern blot hybridization as described (Mago et al., 2004). For this, a PCR amplified sequence from the 5′ end of Sr50 was used as a gene-specific probe. Alternatively, transgene-specific PCR could have been carried out to detect the transgene.
Yeast two-hybrid experiments were performed in Saccharomyces cerevisiae reporter strain Hf7c. The cDNAs encoding full length or truncated Sr50 were cloned at EcoRI-XhoI sites of pGBKT7 and pGADT7 (Clontech). Yeast transformation was performed according to Gietz and Woods (2002) with co-transformants selected on SD media lacking leucine and tryptophan. The interaction analysis was performed by plating the yeast cells on media lacking leucine, tryptophan and histidine and incubating the plates at 30° C. for 3-4 days.
All PCR products used for cloning were generated using Phusion High-Fidelity DNA Polymerase (Finnzymes) with primers listed in Table 2. Molecular cloning was performed using Gateway recombination (Life Technologies) or Quickchange Site-Directed Mutagenesis (Stratagene). For the creation of Gateway entry clones, pDONR207 (Life Technologies) was used. For Agrobacterium tumefaciens (agro) infiltration experiments, pBIN19-35S::GTW:3HA, pBIN19-35S::GTW:CFP (Cesari et al. 2014), pAM-PAT-35s::GTW:YFP:NLS; pAM-PAT-35s::GTW:YFP:nls; pAM-PAT-35s::GTW:YFPv: NES and pAM-PAT-35s::GTW:YFPv:nes were used.
A functional NES from HIV Rev (NES: LQLPPLERLTL; SEQ ID NO:15) and non-functional nes (LQAPPAERATL; SEQ ID NO:16) (Wen et al., 1995) were introduced in the pAM-PAT-35s-GWY-YFPv vector (Bernoux et al., 2008) at the C-terminus end of the YFPv. YFPv-NES/nes fragments were PCR-amplified using a forward primer containing a 3′ SmaI site and a reverse primer containing a 5′ XbaI site as well as the NES or nes sequence. Corresponding PCR products were ligated into pAM-PAT-35s-GWY-YFPv cut with SmaI/XbaI to replace the original YFPv by YFPv-NES/nes fusions.
Transient Protein Expression and Cell Death Assays in N. benthamiana
For Agrobacterium-mediated transformation of N. benthamiana leaf cells, cultures of Agrobacterium strain GV3101 transformed with the genetic construct pMP90 were grown in Luria-Bertani liquid medium containing 50 mg ml-1 rifampicin, 15 mg ml-1 gentamycin and 25 mg ml-1 kanamycin at 28° C. for 24 hours. The constructs providing for expression of NLS-, NES-, nls- and nes-fused proteins were transformed in A. tumefaciens strain GV3103 and grown as described above with addition of 25 mg ml-1 of carbenicillin. Bacteria were harvested by centrifugation, resuspended in infiltration medium (10 mM MES pH 5.6, 10 mM MgCl2 and 150 μM acetosyringone) to an OD600 nm ranging from 0.5 to 1, and incubated for 2 hours at room temperature before leaf infiltration. The infiltrated plants were incubated in growth chambers under controlled conditions for co-immunoprecipitation experiments and cell death assays. For documentation of cell death, leaves were photographed 3-5 days after infiltration.
N. benthamiana epidermal cells were observed under a confocal microscope (TCS SP8; Leica) 20 hours after infiltration. Specific YFP fluorescence was detected using the following spectral settings: excitation, 488 nm; detection, 515-545 nm. Auto-fluorescence of the chloroplasts was detected at 670-730 nm. All images were acquired using a water immersion lens (HC PL APO 63x/1.20 W CORR CS2, Leica).
Protein extraction from N. benthamiana leaves and co-immunoprecipitation experiments were performed as described by Cesari et al. (2014). For immunoblot analysis, proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Pall). Membranes were blocked in 5% skimmed milk and probed with anti-HA or anti-Myc mouse monoclonal antibodies (Roche), followed by goat anti-mouse antibodies conjugated with horseradish peroxidase (Pierce). Labelling was detected using the SuperSignal West Pico or Femto chemiluminescence kits (Pierce). Membranes were stained with Ponceau S to confirm equal loading.
To determine whether the fungal resistance gene Sr50 conferred the same resistance specificity as, or a different resistance specificity to, Sr31, wheat plants of defined lines carrying one or other of these genes, or none of these genes as a control, were tested for their reaction to a set of Pgt strains of different virulence specificities as described in Example 1. Mutant plants carrying mutations in the Sr50 gene were also tested; these were expected to be susceptible to Ptg strains carrying the avirulence gene having Sr50 specificity. The data from the inoculation tests are summarised in Table 3.
The results showed that while Sr31 was, as expected, ineffective in providing resistance to Ptg strains Ug99 (TTKSK) and its derivative TTKST, the Sr50 gene in the genetic background of the wheat variety Gabo provided effective resistance to these strains, yielding an infection type 1 (Table 3). A mutant of Sr50 in this same genetic background (M7 in Table 3) showed a similar phenotype (infection type 2) to the Gabo parent lacking the Sr50 gene, indicating that the mutation in the Sr50 gene in the mutant inactivated the resistance gene. With some inoculations, an intermediate infection phenotype was observed in plants of these lines—this was due to the presence of other stem rust resistance genes in the Gabo background that conferred partial resistance to Ug99 derivatives.
To aid in specifically testing for the Sr50 gene, a spontaneous mutant Pgt strain with virulence for Sr50, designated 98-1,2,3,5,6+Sr50 (available from Plant Breeding Institute accession #130176) was isolated from a single pustule observed after infection of Sr50 plants with strain 98-1,2,3,5,6 (#781219). Infection of a set of differential wheat lines, each containing different R genes, confirmed that the mutant Pgt strain was identical to the parental isolate but with virulence to Sr50. This pair of Pgt strains could therefore be used to specifically detect the presence of Sr50.
The genotypic and phenotypic difference between the Sr31 and Sr50 resistance genes in plants was further confirmed by inoculation of the plants with the parental Ptg and the mutant Ptg derivative, 98-1,2,3,5,6+Sr50. The parental Ptg strain 98-1,2,3,5,6 was virulent on plants of cultivar Gabo, this cultivar having no resistance genes against this Ptg strain, and avirulent on plants containing individually Sr31 or Sr50. In contrast, the mutant Ptg 98-1,2,3,5,6+Sr50 retained avirulence to Sr31 but was virulent to Sr50 (Table 4). Furthermore, the parental strain and the mutant Ptg were both avirulent to plants containing Sr33, a wheat ortholog of Sr50 (see below), as well as to the SrAmigo (1AL.1RS) present in plants of variety Amigo. Therefore, it was concluded that the mutant Sr50 Ptg strain had lost activity of the Sr50 avirulence gene (AvrSr50), and that the Sr50 resistance gene encoded a unique resistance specificity which could be distinguished from the other 1RS located stem rust resistance genes. It was also concluded that Sr50 could be distinguished from the other stem rust resistance genes by inoculation of the plants with different Ptg races.
Mago et al. (2004) observed that a DNA hybridisation probe designated B76, derived from the LRR coding region of the barley powdery mildew resistance gene Mla1, detected at least 17 hybridising fragments in DraI-restricted genomic DNA at the Sr50 locus of wheat and that several Sr50 mutants with small deletions had lost just two of these fragments of about 10 and 12 kbp. In an attempt to isolate the Sr50 gene, a lambda phage genomic DNA library was prepared as described in Example 1 from plants of the Sr50 line Gabo1BL.1RS and screened. The lambda library containing DraI fragments that ranged in size from 9 to 13 kbp. Using the B76 hybridisation probe, six unique clones were isolated from the library and selected for further analysis (
Sequence analysis showed that all of these encoded Mla-related CC-NB-LRR sequences. Three contained various truncations or frameshifts and therefore could not encode full length CC-NB-LRR proteins and two were sequences with some similarity to the LRR region of the B76 probe. The sixth, clone 5 (
To improve physical coverage of the deleted region including Sr50, probe B76 was then used to screen a 1RS chromosome-specific BAC library constructed from a Sr50-containing wheat-rye ditelosomic addition line, as described in Example 1. This probe identified 175 BAC clones. High-information content BAC fingerprinting grouped these into 6 non-overlapping contigs based on 2, 3, 11, 24, 71 and 72 clones. Eighteen BACs forming the minimum tiling path of these contigs were screened by PCR using primers specific to the Mla homolog in lambda clone 5. By this, BAC p2D7 (
To map the Sr50 gene and its surrounding locus onto the physical contig, BACs forming the minimum tiling path from the contig were end-sequenced and PCR markers developed from the BAC ends were used to screen the Sr50 parent and the deletion mutant M2 (00.002). This analysis revealed that the deletion in mutant M2, and therefore the Sr50 gene, was wholly contained in the region spanned by 3 BAC clones, namely p2D7, p2A3 and p2C2, with the proximal and distal ends of p2D7 and p2C2 respectively retained (
These three BAC clones spanning the deletion, as well as BAC p2E7, which was wholly contained in p2D7 and the adjacent BAC, p2C7, were sequenced. Annotation of the nucleotide sequences from this 250 kbp region identified six Mla-related NB-LRR protein coding regions (open reading frames) within the deleted region, and a single NB-LRR ORF in the adjacent region on BAC p2C7 (
They encoded predicted polypeptides in the range of 944 to 974 amino acids. A single copy of a chymotrypsin inhibitor (Ci) gene, a homolog of which was also present at the Mla locus of barley, was also detected within the deleted region. This was consistent with previous DNA hybridization analyses which identified 4 copies of a related Ci gene on 1RS, only one of which was deleted in the interstitial deletion mutants (Mago et al., 2004). The amino acid sequence encoded by the lambda clone 5 was 100% identical to the ScRGA1-A amino acid sequence, but the other lambda clones identified as described above did not correspond to any of the other ScRGA1 genes in the BAC contig.
In addition to the M2 deletion mutant described above, the inventors had previously isolated several EMS-derived Sr50 gene mutants that retained all of the B76-hybridising fragments and therefore possibly represented point mutations of Sr50 (Mago et al. 2004). Two mutant plants, M7 (00.007) and M13 (01.013), were recovered and progeny plants produced. A cross between M7 and M13 plants produced no resistant progeny indicating they carried a mutation in the same gene. The six ScRGA1 genes identified within the M2 deletion as described above were amplified from M7 and from M13 as well as from Gabo1DL.1RS DR.A1 and sequenced. One gene candidate of the six, namely ScRGA1-A, contained a single base pair deletion in M7 and a 23 bp deletion in M13. Both mutations resulted in translational frame shifts which led to premature stop codons in the ORF (
To confirm that ScRGA1-A conferred Sr50 resistance, two constructs containing this gene were used to transform plants of the stem rust-susceptible wheat cultivar Fielder as described in Example 1. The first construct, pVecBarSr50 (SEQ ID NO: 12), contained a 7.9 kb PCR-amplified genomic sequence of ScRGA1-A including the native promoter and terminator (2.4 and 1.4 kbp, respectively) within the T-DNA. The second construct, pVecNeoSr50 (SEQ ID NO: 11), contained a 9.7 kb NotI genomic fragment from BAC p2D7 with larger 5′ and 3′ regions, within the T-DNA. Both constructs therefore contained the genomic form of ScRGA1-A including all of its introns as well as the native promoter, but linked to heterologous sequences within a chimeric T-DNA construct. The constructs were used to transform wheat and confirmed transgenic plants and their progeny tested as described in Example 1.
Independent T0 transgenic plants and T1 progeny plants containing the T-DNA from pVecBarSr50 were inoculated with Pgt strains. The transgenic plants exhibited responses to Pgt typical of Sr50-mediated resistance whereas segregant plants that lacked the transgene were as susceptible as untransformed parental plants. Likewise, TO wheat lines transformed with pVecNeoSr50 showed resistance typical of Sr50. To confirm that the observed resistance phenotype was race specific and not due to general enhancement of defense pathways, T1 progeny of four stem rust resistant transgenic plants (lines 2, 10b, 13 and 19d) were tested for their reactions to leaf rust and stripe rust by inoculation with the strains described in Example 1.
All transgenic progeny plants were as susceptible to leaf rust and stripe rust as the non-transformed controls. Moreover, the T1 progeny populations showed a 3:1 segregation ratio (resistant:susceptible) for reaction to stem rust, indicative of Mendelian inheritance ratios for insertion of a single transgene in the nuclear genome. The combined mutational and complementation data conclusively demonstrated that the gene designated ScRGA1-A was the Sr50 resistance gene.
The experiment also proved that the gene provided rust resistance when used to transform a cereal plant. Thus, as the skilled person would appreciate, the same or similar constructs (for example with different promoter regions) can be used to produce other cereal plants (such as rice, barley, maize or sorghum) which express Sr50 resulting in resistance to one or more races Puccinia graminis.
The presence of two predicted introns in the protein coding region of Sr50 was confirmed by RT-PCR and comparison of the cDNA and genomic nucleotide sequences. Rapid amplification of cDNA ends (RACE) analysis, carried out as described by Jordan et al. (2011), identified 5′ and 3′ UTRs of 629 and 238 bp, respectively, containing 3 additional introns (
The predicted Sr50 polypeptide was 77-78% identical in amino acid sequence to polypeptides in the barley MLA protein family, some of which confer resistance to powdery mildew, not rust resistance. Phylogenetic analysis of cereal Mla homologs using their amino acid sequences placed the Sr50 protein in a clade with other ScRGA1 polypeptides as well as with the orthologous proteins TmMla1 and Sr33 from diploid wheat species, T. monococcum and Ae. tauschii (
To detect the presence of the Sr50 gene in rye, the origin species of the Sr50 gene in wheat, 114 geographically diverse rye accessions were screened by PCR with primers flanking Sr50. The PCR amplification was done using PfuUltra II Fusion HS DNA Polymerase and oligonucleotide primers F1-R1 followed by nested PCR with primers F3-R3. These reactions amplified a 4.17 kb PCR product including the entire Sr50 gene.
Amplification of Sr50 sequences occurred from only 10 of the 114 accessions, indicating the presence of a Sr50 gene or a close homolog in those 10 accessions. For five of those accessions, the amplified gene was identical to Sr50, while three of the others contained small substitutions and two were disrupted by a sequence inversion. All of these rye accessions except for Dwarf Petkus R1 showed resistance to several races of stem rust, including the Sr50-virulent mutant, indicating the presence of additional Sr genes, which may be on other chromosomes. Despite the sequence similarity of the Sr50 gene in most of these accessions, they contained diverse haplotypes of this complex locus, suggesting extensive recombination within this cluster in rye. Both of the Sr31 and stripe rust resistance Yr9 genes occur in the same 1RS region, and may also belong to this gene family Therefore, this locus appears to be a hotspot for evolution of fungal resistance specificities in cereals, including in wheat, barley and rye.
Previous functional analysis of the MLA N-terminal region showed that a 225 amino acid fragment including the CC and part of the NB domains could self-associate in yeast, whereas a 160 amino acid fragment containing the CC domain alone was autoactive in signalling cell death in planta. A 120 amino acid fragment, which was a truncated CC domain, could dimerise in vitro (Maekawa et al., 2011; Bai et al., 2012).
To investigate whether the wheat and rye orthologs Sr33 and Sr50 functioned similarly to MLA and to determine the minimal functional region in cell death signalling, various N-terminal fragments of the Sr50 and Sr33 polypeptides were fused to either a C-terminal HA or CFP tag. These fusion polypeptides were transiently expressed in N. benthamiana leaf cells under the control of the 35S promoter as described in Example 1. The constructs that expressed the full CC domains of Sr33 and Sr50, namely amino acids 1-160 and 1-163, respectively, triggered a strong cell death response in the leaf cells that was visible 40 hours after agro-infiltration as a leaf tissue necrosis of the infiltrated zones.
Similar to previous reports (Makeawa et al., 2011; Bai et al., 2012), the corresponding domain form barley MLA, MLA101-160, also induced a strong cell death response that was visible within 24 hours when expressed in N. benthamiana leaf cells. Another construct made to express a positive control protein fusion, the rice autoactive CC-NB-LRR RGA4 (Cesari et al., 2014), also produced a strong cell death response, while a mutant variant of RGA4 did not cause a response in N. benthamiana leaf cells. Expression of the CC domains together with a portion of the NB domains of Sr33 (1-225), Sr50 (1-228) and MLA10 (1-225) also triggered strong cell death responses. However, the truncated CC domains of MLA10 (1-120), Sr33 (1-120) and Sr50 (1-123) fused to the HA tag did not induce cell death in N. benthamiana. Western blot analysis showed that all fusion proteins were properly expressed in the leaf cells.
Taken together, these results showed that the full CC domains of MLA10, Sr33 and Sr50 were required and sufficient for induction of cell death signalling, and although the truncated CC domain of MLA10 dimerized in solution and its crystal structure had been resolved (Maekawa et al., 2011), it was not sufficient to trigger a cell death response. These data demonstrated that the function of the CC domain in each polypeptide was to trigger the induction of cell death, which is associated with the resistance response in wheat stem cells when the pathogen is present.
Although the truncated MLA10 CC5-120 fragment was able to self-associate in solution to form a dimer, only the MLA10 CC-NB1-225 domain had been shown to self-interact when tested in a yeast two-hybrid assay (Maekawa et al., 2011). Self-interaction had not been demonstrated with the minimal active CC1-160 domain. Therefore, yeast-two-hybrid experiments were carried out to test for interaction between the truncated CC, CC and CC-NB fragments of MLA10 and Sr50 Immunoblotting showed that all the protein fragments were expressed in the yeast cells in the experiments. The results showed that the full CC domains of MLA10 and Sr50 were required for self association in yeast, whereas no interaction was detected for the truncated CC domains of these proteins.
To determine whether the active CC domains of MLA10, Sr33 and Sr50 self-associated in planta, combinations of the HA and CFP-tagged MLA10, Sr33 and Sr50 domains were co-expressed in N. benthamiana leaf cells and co-immunoprecipitation assays were performed. The CC1-284 domain of the unrelated Lr21 resistance polypeptide, which was known to be autoactive in planta, was also included in the experiment to test its ability to self-interact. The RGA4 CC domain (amino acids 1-171) fused to CFP was used as a control for binding specificity Immunoblotting using anti-GFP and anti-HA antibodies showed that all of the polypeptides were expressed as intended, except for the MLA10 fusions, which was likely due to the more rapid cell death response induced by these polypeptides and their consequent degradation in the cells Immunoprecipitation was used to assay for association of the co-expressed polypeptides Immunoprecipitation with anti-GFP antibodies resulted in enrichment of all CFP-fused proteins and Sr331-160:HA, Sr501-163:HA and Lr211-284:HA specifically co-precipitated with Sr331-160:CFP, Sr501-163:CFP and Lr211-284:CFP, respectively, but not with RGA41-171:CFP. Taken together, these results showed that the CC domains of Sr33, Sr50 and Lr21 had the capability to form specific homo-complexes in planta, i.e. the could self-associate.
The MLA10 full length polypeptide and its CC-NB1-225 domain have been shown to trigger cell-death signalling when localised in the cytoplasm of N. benthamiana leaf cells (Bai et al., 2012). To determine whether the shorter MLA10 CC1-160 domain, thought to be needed for cell death induction, by itself could induce cell death when localised in the cytoplasm and to test this feature for the Sr33 and Sr50 CC domains, those domains were transiently expressed in N. benthamiana fused to YFP along with a nuclear localisation signal (NLS), a mutated NLS (nls), a nuclear export signal (NES) or a mutated NES (nes). Upon expression of the YFP:NLS fused CC domains, specific YFP fluorescence was detected exclusively in the nuclei of N. benthamiana cells. In contrast, the CC domains fused to YFP:NES were effectively excluded from the nuclei, with YFP fluorescence detected in the cytosol and surrounding the nuclei only, while the mutated nls and nes variants allowed detection of YFP fluorescence in both the cytosol and the nuclei as intended. Cell death assays in N. benthamiana leaf cells revealed that forced nuclear localization of all CC domains reduced their cell-death activity. Other fusions with YFP:NES, YFP:nes or YFP:nls did not affect the cell-death inducing activity of MLA10 or Sr33 CC domains. In the case of the Sr50 CC domain, the mutant nls also showed some reduction in cell death activity relative to the NES and nes fusions. Protein expression of all constructs was verified by immunoblotting. The NES fusion constructs consistently showed lower protein accumulation. Nevertheless, these constructs gave the strongest cell death (hypersensitive response, HR) phenotype, suggesting that cell death and protein degradation processes were already activated at the time of sampling. These results indicated that cytosolic localization of those CC domains was required for cell death induction.
The Mla gene clade of CC-NB-LRR genes, originally identified on chromosome 1HS in barley as a powdery mildew resistance gene also occurs in wheat, where orthologs include the powdery mildew resistance gene TmMla1 (Jordon et al., 2011) and the stem rust resistance gene Sr33 (Periyannan et al., 2013). The genes occur as a small gene family of about 5 members in wheat and barley. DNA gel blot analysis has shown that this family has greatly expanded to over 20 members on chromosome IRS in rye (Mago et al., 2004). The experiments described above have now shown that the Sr50 stem rust resistance gene is a member of the rye Mla orthologous gene family
Although the wheat stem rust resistance genes Sr33 and Sr35 were isolated previously (Periyannan et al., 2013; Saintenac et al., 2013), originating from wheat, the Sr50 gene was the first stem rust resistance gene to be cloned which originated from rye and which is effective against all known field races of Pgt including Ug99. High resolution mapping and mutation studies have also placed both Sr31 and the stripe rust resistance gene Yr9 from Petkus rye IRS in the region homoeologous to Sr50 (Mago et al., 2005), suggesting that these genes may also belong to the Mla gene family Therefore, this multi-species locus appears to be a hotspot for evolution of resistance specificities in the cereals.
Increasing knowledge about localization of NB-LRR proteins has revealed that they can be observed and activated in a wide diversity of cellular compartments. Some are exclusively nuclear such as RRS1 (Deslandes et al., 2003; Tasset et al., 2010), whereas others like RGA4 and RGA5 are mainly localized in the cytosol (Cesari et al., 2014). Rpm1 (Gao et al., 2011), RPS2 (Axtell and Staskawicz, 2003) and RPS5 (Qi et al., 2012) are located at the plasma membrane, and the flax L6 and M proteins occur on the Golgi and tonoplast membranes, respectively (Takemoto et al., 2012). However, many show a nucleo-cytoplasmic localization such as N (Burch-Smith et al., 2007; Caplan et al., 2008), RPS4 (Wirthmueller et al., 2007), SNC1 (Cheng et al., 2009), Pik1/Pik2 (Zhai et al., 2014), Rx (Slootweg et al., 2010) and MLA10 (Shen et al., 2007).
In the case of the potato CC-NB-LRR polypeptide Rx that triggers recognition of the Potato Virus X (PVX) coat protein (CP), it was shown that CP activates Rx in the cytoplasm, and forced nuclear localization of Rx severely compromised both virus resistance and cell death (Slootweg et al., 2010; Tameling et al., 2010). However, Rx nuclear exclusion only moderately reduced PVX resistance (Slootweg et al., 2010), suggesting that the cytosolic pool was most important, although both nuclear and cytoplasmic pools of the resistance protein may have contributed to resistance signalling. Conversely, another study on MLA10 demonstrated that a nuclear localised receptor was sufficient to confer resistance to B. graminis in a transient single cell assay in barley, while a nuclear-excluded MLA10 was not (Shen et al., 2007). However, forced nuclear localisation of autoactive MLA protein variants impaired induction of cell death whereas nuclear excluded variants were sufficient to trigger resistance (Bai et al., 2012). To explain this, it has been proposed that MLA10-triggered cell death signalling and disease resistance signalling occur in different compartments (Bai et al., 2012). Alternatively, AVRA10 recognition may occur exclusively in the nucleus, explaining the requirement for a nuclear localised receptor to confer resistance, with subsequent signalling events perhaps occurring in the cytoplasm.
Sr33 and, as demonstrated by the experiments described above, Sr50 are homologs of MLA10 and using their minimal CC signaling domains, a similar pattern of cell death induction was observed relative to that triggered by the full length MLA10 protein or the MLA10 CC-NB domain (Bai et al., 2012). Therefore, it appeared that the responses triggered by MLA10 in barley and its close wheat homologs were similar, suggesting a conserved cell death pathway triggered by different pathogens in two different cereal species.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
This application claims priority from AU 2015904976 filed 1 Dec. 2015, the entire contents of which are incorporated herein by reference.
All publications discussed and/or referenced herein are incorporated herein in their entirety.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2015904976 | Dec 2015 | AU | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15779782 | May 2018 | US |
| Child | 18087162 | US |
