Patent Application
20200291419
The present invention is directed to the field of plant genetics and wheat and cereal crop breeding.
Stripe rust (yellow rust), caused by the pathogen Puccinia striiformis f. sp. tritici (Pst), is a devastating fungal disease of wheat, causing severe yield losses mainly due to new virulent races that have appeared within the past decades. The use of race-specific resistance genes in wheat varieties has been considered as the most efficient method for disease management. However, the rapid evolution of resistance-breaking pathogen races is emphasizing the need for new sources of broad-spectrum resistance to stripe rust. The genetic bottlenecks associated with plant domestication and subsequent selection in manmade agroecosystems have reduced the genetic diversity of modern crops and increased their vulnerability to biotic and abiotic stresses. One of the ways to address this problem is to recruit the adaptive potential of the wild germplasm. Wild emmer wheat, the tetraploid progenitor of domesticated wheat, distributed along a wide range of habitats in the Fertile Crescent, has valuable unexploited adaptive diversity to various diseases, including stripe rust.
Yr15 is a broad-spectrum stripe rust resistance gene discovered in the 1980s in wild emmer wheat Triticum turgidum ssp. dicoccoides (DIC hereafter) accession G25 (G25, hereafter). Since its discovery, Yr15 has been introgressed into a variety of worldwide durum and bread wheat genetic backgrounds and has provided robust protection against a wide range of Pst isolates from over 34 countries around the world. Previously, Yr15 was mapped to the short arm of chromosome 1B, and for example, a recombinant inbred family segregating for Yr15 was generated by crossing resistant introgression durum lines (B9, B10), which carry Yr15 from DIC G25, with the susceptible recurrent line D447. Importantly, Yr15 has been introduced from wild wheat into cultivated durum and common wheat to new species only by introgression. The full sequence of the gene was not known, and thus vectors for introduction of the gene do not exist. There is a great need for compositions and methods for conferring Pst resistance to new species. Additionally, resistance genes that can combat the new and more virulent races of Pst are essential.
The present invention provides isolated DNA of the Wtk1 gene or a functional equivalent capable of conferring resistance to stripe rust, as well as artificial vectors comprising same and proteins encoded by same. Transgenic plants, as well as cells, seeds, and tissue therefrom which express the Wtk1 gene or a functional equivalent thereof are also provided.
According to a first aspect, there is provided an isolated DNA comprising a nucleic acid sequence with at least 80% homology to SEQ ID NO: 1 or a fragment thereof that encodes a protein that confers resistance to Stripe Rust (Pst).
According to another aspect, there is provided an artificial vector comprising the isolated DNA of the invention.
According to another aspect, there is provided a transgenic plant or cell thereof, comprising an isolated DNA of the invention or an artificial vector of the invention.
According to another aspect, there is provided an isolated protein encoded by an isolated DNA of the invention.
According to another aspect, there is provided a method of conferring resistance to Pst to a plant or cell thereof, the method comprising at least one of:
thereby conferring resistance to Pst to a plant or cell thereof.
According to another aspect, there is provided a nucleic acid molecule capable of binding differentially to a functional Wtk1 nucleic acid molecule and a non-functional wtk1 nucleic acid molecule, wherein a functional Wtk1 confers resistance to stripe rust (Pst).
According to another aspect, there is provided a method for detecting a functional Wtk1 gene in a sample, wherein a functional Wtk1 confers resistance to stripe rust (Pst), comprising:
thereby, detecting a functional Wtk1 gene in a sample.
According to another aspect, there is provided a kit comprising at least 2 nucleic acid molecules, wherein the molecules comprise a sequence selected from SEQ ID NO: 69-70 and 103-110.
According to another aspect, there is provided a method of conferring resistance to a pathogen to a plant or a cell, the method comprising expressing in the plant or cell thereof at least protein comprising an amino acid sequence selected from SEQ ID NOs: 124-214.
According to some embodiments, the DNA encodes a protein that confers resistance to Pst. According to some embodiments, the protein that confers resistance comprises the amino acid sequence of SEQ ID NO: 2;
According to some embodiments, the nucleic acid sequence comprises a sequence with at least 80% homology to SEQ ID NO: 6 and a sequence with at least 80% homology to SEQ ID NO: 7. According to some embodiments, the nucleic acid sequence encodes for a first kinase-like domain of Wtk1 (KinI, SEQ ID NO: 4) and a second kinase-like domain of Wtk1 (KinII, SEQ ID NO: 5).
According to some embodiments, the Pst comprises the Warrior race of Pst.
According to some embodiments, the nucleic acid sequence has at least 95% homology to SEQ ID NO: 1. According to some embodiments, the nucleic acid sequence comprises SEQ ID NO: 1. According to some embodiments, the nucleic acid sequence consists of SEQ ID NO: 1.
According to some embodiments, the vector of the invention further comprises at least one nucleic acid sequence with at least 80% homology to a tandem kinase-pseudokinase (TKP)-containing gene. According to some embodiments, the nucleic acid sequence with at least 80% homology to a TKP-containing gene encodes an amino acid sequence selected from SEQ ID NOs: 123-214.
According to some embodiments, the vector of the invention further comprises at least one nucleic acid sequence of a pathogen-resistance gene. According to some embodiments, the pathogen is Pst. According to some embodiments, the pathogen-resistance gene is selected from Yr36, Yr5, Yr18 and Yr46. According to some embodiments, the pathogen-resistance gene is Yr5.
According to some embodiments, the vector of the invention comprises at least one promoter for transcription in plant cells. According to some embodiments, the promoter is the endogenous Wtk1 promoter. According to some embodiments, the at least one promoter is operably linked to the isolated DNA. According to some embodiments, the vector of the invention comprises at least another promoter for transcription in plants operably linked to a TKP-containing gene or a pathogen resistance gene.
According to some embodiments, the sample is from a plant. According to some embodiments, the plant is a cereal plant. According to some embodiments, the cereal plant is any one of barley, rye, triticale, oat, wheat, rice and maize. According to some embodiments, the cereal plant is wheat.
According to some embodiments, the artificial vector of the invention and/or the isolated DNA of the invention is for use in conferring resistance to Pst to a cell of a plant.
According to some embodiments, the isolated protein of the invention comprises an amino acid sequence with at least 80% homology to SEQ ID NO: 2, wherein the isolated protein confers resistance to Pst. According to some embodiments, the isolated protein of the invention consists of the amino acid sequence of SEQ ID NO: 2.
According to some embodiments, the functional Wtk1 nucleic acid molecule is genomic DNA and comprises a nucleic acid sequence comprising SEQ ID NO: 3. According to some embodiments, the functional Wtk1 nucleic acid molecule is cDNA synthesized from mRNA and comprises a nucleic acid sequence comprising SEQ ID NO: 1. According to some embodiments, the non-functional Wtk1 nucleic acid molecule is cDNA synthesized from mRNA and comprises a nucleic acid sequence comprising any one of SEQ ID NOs: 111-117.
According to some embodiments, binding differentially comprises 100% complementarity to the functional Wtk1 nucleic acid molecule or to the non-functional Wtk1 nucleic acid molecule, but not to both.
According to some embodiments, the nucleic acid molecule comprises at least one single nucleotide polymorphism (SNP) that differentiates between a functional Wtk1 nucleic acid molecule and a non-functional wtk1 nucleic acid molecule. According to some embodiments, detecting comprises detecting at least one single nucleotide polymorphism (SNP) that differentiates between SEQ ID NO: 1 and a non-functional wtk1 nucleic acid molecule. According to some embodiments, the SNP is selected from Table 15.
According to some embodiments, the nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 69-70 and 103-110. According to some embodiments, the nucleic acid molecule consists of the sequence of any one of SEQ ID NO: 69-70 and 103-110.
According to some embodiments, the nucleic acid molecule is a primer. According to some embodiments, the nucleic acid molecule comprises a tag.
According to some embodiments, the detecting comprises:
According to some embodiments, the at least one nucleic acid molecule comprises a tag and the detecting the hybridizing comprises detecting the tag.
According to some embodiments, the hybridizing comprises at least one of, polymerase chain reaction (PCR), southern blotting and northern blotting.
According to some embodiments, the PCR comprises:
According to some embodiments, the primers are Kompetative Allele Specific PCR (KASP) primers.
According to some embodiments, the method of the invention comprises amplifying the DNA with a primer comprising the sequence of SEQ ID NO: 105, a primer comprising the sequence of SEQ ID NO: 106, a primer comprising the sequence of SEQ ID NO: 107, a primer comprising the sequence of SEQ ID NO: 108, a primer comprising the sequence of SEQ ID NO: 109, and a primer comprising the sequence of SEQ ID NO: 110.
According to some embodiments, the kit of the invention comprises a molecule comprising the sequence of SEQ ID NO: 105, a molecule comprising the sequence of SEQ ID NO: 107, and optionally a molecule comprising the sequence of SEQ ID NO: 106. According to some embodiments, the kit of the invention comprises the sequence of SEQ ID NO: 108, a molecule comprising the sequence of SEQ ID NO: 110, and optionally a molecule comprising the sequence of SEQ ID NO: 109.
According to some embodiments, at least one of the molecules comprises a tag.
Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description together with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
The present invention provides isolated DNA of the Wtk1 gene or a functional equivalent capable of conferring resistance to stripe ruse (Pst). Artificial vectors comprising same and proteins encoded by same are also provided, as are transgenic plants, and plant tissues and parts therefore which express Wtk1 or a functional equivalent.
The present invention further provides nucleic acid molecules and primers, as well as methods of use thereof, for differentiating between functional Wtk1 and non-functional WtK1, wherein functional Wtk1 confers resistance to Pst.
The present invention is based, in part, on the surprising finding that the genomic region containing the predicted Yr15 gene, which confers stripe rust-resistance, contains an open region frame for a Wheat Tandem Kinase (Wtk1) gene, which encodes a novel non-membrane-bound tandem kinase protein (WTK1) which actually confers the resistance. Further, this protein confers a broad resistance to Pst strains, including many of the new highly virulent forms of Pst. Both kinase domains are required for this resistance, and multiple wtk1 non-functional alleles with mutations in the coding region lack a Pst-resistance phenotype and are found in many domesticated species that are susceptible to Pst. Furthermore, the primers of the invention are able to distinguish between these non-functional alleles and/or pseudogenes and the functional Wtk1.
As used herein, “Pst” refers to stripe rust, which is also called Puccinia striiformis f sp. tritici. In some embodiments, Pst comprises all strains of stripe rust. In some embodiments, Pst comprises European stripe rust. In some embodiments, Pst comprises the Warrior race (e.g. DK09/11) of stripe rust.
As used herein, “Wtk1” refers to a functional Wtk1 gene, which confers resistance to Pst. As used herein, “wtk1” refers to a non-functional wtk1 gene, which does not confer resistance to Pst. The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. Thus, the term “gene” includes coding sequences (CDS) and/or regulatory sequences required for expression. The term “gene” can also apply to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
In some embodiments, the functional Wtk1 gene comprises the Wtk1 CDS. In some embodiments, the Wtk1 CDS consists of the nucleic acid sequence
In some embodiments, the Wtk1 CDS comprises SEQ ID NO: 1.
In some embodiments, the functional Wtk1 gene comprises the Wtk1 genomic sequence which consists of the nucleic acid sequence
In some embodiments, the Wtk1 genomic sequence comprises SEQ ID NO:3. In some embodiments, the Wtk1 genomic sequence comprises the genomic sequence of Wtk1 and 2 kilobases upstream and/or downstream from that sequence.
In some embodiments, the Wtk1 CDS is a cDNA reverse transcribed from an RNA which codes for the amino acid sequence
In some embodiments, the Wtk1 CDS is a cDNA reverse-transcribed from an RNA which codes for a polypeptide comprising a first kinase-like domain (KinI) of Wtk1 and a nucleic acid sequence that encodes a second kinase-like domain (KinII) of Wtk1. Each possibility represents a separate embodiment of the invention. In some embodiments, the KinI consists of the amino acid sequence
In some embodiments, the KinII consists of the amino acid sequence
As used herein, the terms “peptide”, “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms “peptide”, “polypeptide” and “protein” as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides, polypeptides and proteins described have modifications rendering them more stable while in the organism or more capable of penetrating into cells. In one embodiment, the terms “peptide”, “polypeptide” and “protein” apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide”, “polypeptide” and “protein” apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.
By a first aspect there is provided an isolated DNA comprising a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to the Wtk1 coding sequence. Each possibility represents a separate embodiment of the invention.
By another aspect, there is provided an isolated DNA comprising a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% identity to the Wtk1 coding sequence. Each possibility represents a separate embodiment of the invention.
In some embodiments, the isolated DNA is at least 100%, 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% pure. Each possibility represents a separate embodiment of the invention. The purity is with respect to contamination by other DNAs or other cellular components.
In some embodiments, the isolated DNA is a cDNA. In some embodiments, the isolated DNA is an isolated Wtk1 CDNA. In some embodiments, the isolated DNA of the invention comprises a nucleic acid sequence with at least 80% homology to SEQ ID NO: 1. In some embodiments, the isolated DNA of the invention comprises SEQ ID NO: 1.
In some embodiments, the isolated DNA of the invention comprises a fragment or an analog to the Wtk1 gene, wherein the fragment or analog encodes a protein that confers resistance to Pst. In some embodiments, the isolated DNA of the invention comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to a fragment or analog of SEQ ID NO: 1, wherein the fragment encodes a protein that confers resistance to Pst. Each possibility represents a separate embodiment of the invention. In some embodiments, the isolated DNA molecules of the invention encode for a protein that confers resistance to Pst.
In some embodiments, the isolated DNA of the invention comprises a nucleic acid sequence that encodes a peptide comprising SEQ ID NO: 2. In some embodiments, the isolated DNA of the invention comprises a nucleic acid sequence that encodes a peptide with at least 99%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO: 2 and confers resistance to Pst. Each possibility represents a separate embodiment of the invention.
In some embodiments, the isolated DNA comprises the genomic sequence of Wtk1. In some embodiments, the genomic sequence of Wtk1 comprises SEQ ID NO: 3. In some embodiments, the isolated DNA comprises the genomic sequence of Wtk1 and 2 kilobases (kb) upstream and/or downstream from that sequence. In some embodiments, the isolated DNA comprises regulatory element that control the expression of Wtk1. In some embodiments, the isolated DNA comprises the genomic sequence 3.4 kb upstream and 1 kb downstream of the sequence provided in SEQ ID NO: 3. In some embodiments, the isolated DNA comprises the sequence provided in SEQ ID NO: 117.
As used herein, the term “isolated nucleic acid molecule” refers to a nucleic acid molecule that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the nucleic acid in nature. Typically, a preparation of isolated DNA or RNA contains the nucleic acid in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. In some embodiments, the isolated DNA is cDNA. In some embodiments, the isolated nucleic acid is any one of DNA, RNA, and cDNA. In some embodiments, the isolated nucleic acid molecule is a synthesized nucleic acid molecule. Synthesis of nucleic acid molecules is well known in the art and may be performed, for example, by ligating or covalently linking by primer linkers multiple nucleic acid molecules together.
The term “nucleic acid” is well known in the art. A “nucleic acid” as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising nucleotides. Nucleotides are comprised of nucleosides and phosphate groups. The nitrogenous bases of nucleosides include, for example, naturally occurring purine or pyrimidine nucleosides as found in DNA (e.g., an adenine “A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” or a C).
The term “nucleic acid molecule” includes but not limited to single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), small RNAs, circular nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, amplification products, modified nucleic acids, plasmid or organellar nucleic acids, and artificial nucleic acids such as oligonucleotides.
As used herein, the term “analog” includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities to confer resistance to Pst. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention.
In some embodiments, the isolated DNA of the invention comprises a nucleic acid sequence that encodes a first kinase-like domain (KinI) of Wtk1 and a nucleic acid sequence that encodes a second kinase-like domain (KinII) of Wtk1. Each possibility represents a separate embodiment of the invention. In some embodiments, the KinI consists of SEQ ID NO: 4. In some embodiments, the KinII consists of SEQ ID NO: 5. In some embodiments, the isolated DNA comprises a nucleic acid sequence encoding SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, the DNA encoding SEQ ID NO: 4 and SEQ ID NO:5 are separated by a spacer. In some embodiments, the spacer is at least 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 base pairs. Each possibility represents a separate embodiment of the invention. In some embodiments, the spacer has a nucleic acid bases in a multiple of 3 such that the KinII will be encoded in frame.
In some embodiments, the isolated DNA of the invention comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to DNA encoding KinI of Wtk1 and a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to the DNA encoding KinII of Wtk1. Each possibility represents a separate embodiment of the invention. In some embodiments, the DNA encoding KinI of Wtk1 is
In some embodiments, the DNA encoding KinII of Wtk1 is
In some embodiments, the isolated DNA of the invention comprises a nucleic acid sequence with at least 80% homology to SEQ ID NO: 6 and a nucleic acid sequence with at least 80% homology to SEQ ID NO: 7. In some embodiments, the isolated DNA comprises a nucleic acid sequence that encodes a protein that confers resistance to Pst. In some embodiments, the isolated DNA comprises a nucleic acid sequence with at least 95% homology to SEQ ID NO: 1. In some embodiments, the isolated DNA consists of the nucleic acid sequence of SEQ ID NO: 1.
By another aspect there is provided an artificial vector comprising the isolated DNA of the invention. In some embodiments, the artificial vector is an expression vector. In some embodiments, the artificial vector is a plant expression vector. In some embodiments, the artificial vector is for use in expressing Wtk1. In some embodiments, the artificial vector is for use in expressing Wtk1 in a plant or cell thereof. In some embodiments, the artificial vector is for use in conferring resistance to Pst. In some embodiments, the artificial vector is for use in conferring resistance to Pst to a plant or cell thereof.
Expressing of a gene within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell's genome. In some embodiments, the gene is in an expression vector such as plasmid or viral vector. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector, a virgaviridae viral vector, or a poxviral vector. The barley stripe mosaic virus (BSMV), the tobacco rattle virus and the cabbage leaf curl geminivirus (CbLCV) may also be used. The promoters may be active in plant cells. The promoters may be a viral promoter.
In some embodiments, the gene is operably linked to a promoter. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). In some embodiments, the promoter is operably linked to an isolated DNA of the invention. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is the endogenous promoter. In some embodiments, the endogenous promoter is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 kb upstream of the transcriptional start site of Wtk1. Each possibility represents a separate embodiment of the invention. In some embodiments, the endogenous promoter comprises SEQ ID NO: 118.
In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), such as biolistic use of coated particles, and needle-like particles, Agrobacterium Ti plasmids and/or the like.
The term “promoter” as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. The promoter may extend upstream or downstream of the transcriptional start site, and may be any size ranging from a few base pairs to several kilo-bases.
In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.
In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.
In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
In some embodiments, recombinant viral vectors, which offer advantages such as systemic infection and targeting specificity, are used for in vivo expression. In one embodiment, systemic infection is inherent in the life cycle of, for example, the retrovirus and is the process by which a single infected cell produces many progeny virions that infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread systemically. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
In some embodiments, plant viral vectors are used. In some embodiments, a wild-type virus is used. In some embodiments, a deconstructed virus such as are known in the art is used. In some embodiments, Agrobacterium is used to introduce the vector of the invention into a virus.
Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, agrobacterium Ti plasmids and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield, or activity of the expressed polypeptide.
In some embodiments, the artificial vector further comprises at least one nucleic acid sequence of a tandem kinase-pseudokinase (TKP)-containing gene. In some embodiments, the artificial vector further comprises at least one nucleic acid sequence of a pathogen resistance gene.
In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a tandem kinase-pseudokinase (TKP)-containing gene. Each possibility represents a separate embodiment of the invention. As used herein a TKP-containing gene is a gene with an active kinase and inactive pseudokinse domain in tandem. In some embodiments, the TKP-containing gene comprises no other protein motifs. In some embodiments, the TKP-domain is homologous to the TKP domain of Wtk1. In some embodiments, the TKP-containing gene is selected from the genes provided in
In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a pathogen-resistance gene. As used herein, the term “pathogen-resistance gene” refers to a gene that provides a plant or plant cell with resistance to a pathogen. In some embodiments, the pathogen is a bacterial pathogen. In some embodiments, pathogen is Pst. Pathogen-resistance genes are well known in the art, and include, but are not limited to Yr18, Yr5, Yr36, Yr46, Yr17, Yr29, Lr10, Lr13, Yr58, Sr13 and Sr 21. Yr18 is also known as Lr34 and Sr57. Yr29 is also known as Lr46. Yr46 is also known as Lr67. Yr18 is also known as Lr34 and Sr57. In some embodiments, the pathogen resistance gene is selected from Yr18, Yr5, Yr36, Yr46, Yr17, Yr29, Lr10, Lr13, Yr58, Sr13 and Sr 21. In some embodiments, the pathogen resistance gene is selected from Yr18, Yr5, Yr36, and Yr46. In some embodiments, the pathogen resistance gene is selected from Yr18, Yr5, and Yr36. In some embodiments, the pathogen resistance gene is selected from Yr18, and Yr5. In some embodiments, the pathogen resistance gene is Yr5.
In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yr36 gene. Each possibility represents a separate embodiment of the invention. In some embodiments, Yr36 comprises any one of the nucleic acid sequences denoted by SEQ ID NOs: 8-13. In so embodiments, the artificial vector comprises a Yr36 gene. In some embodiments, the artificial vector comprises any one of SEQ ID NOs: 8-13. In some embodiments, the Yr36 gene is operably linked to a plant promoter. In some embodiments, the artificial vector comprises a nucleic acid sequence that encodes for a Yr36 protein. In some embodiments, the Yr36 protein has the amino acid sequence denoted by any one of SEQ ID NO: 14-18.
In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yr5 gene. Each possibility represents a separate embodiment of the invention. In some embodiments, the artificial vector further comprises a Yr5 gene. In some embodiments, the Yr5 gene is operably linked to a plant promoter. In some embodiments, the artificial vector further comprises a nucleic acid sequence that encodes for a Yr5 protein. In some embodiments, the Yr5 genes encodes a protein comprising the sequence
In some embodiments, the sequence of Yr5 comprises a sequence selected from the sequences provided in Accession numbers JQ318576.1, JQ318577.1, JQ318578.1, JQ318579.1, JQ318580.1, JQ318581.1, JQ318582.1, JQ318583.1, JQ318584.1, JQ318585.1, JQ318586.1, JQ318587.1, and JQ318588.1.
In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yr18 gene. Each possibility represents a separate embodiment of the invention. In so embodiments, the artificial vector further comprises a Yr18 gene. In some embodiments, the Yr18 gene is operably linked to a plant promoter. In some embodiments, the artificial vector further comprises a nucleic acid sequence that encodes for a Yr18 protein. In some embodiments, the Yr18 gene encodes a protein with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to the sequence
Each possibility represents a separate embodiment of the invention. In some embodiments, the Yr18 gene comprises a nucleotide sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to the sequence provided in accession number XM_015795636.1. In some embodiments, the sequence of Yr5 comprises a sequence selected from the sequences provided in Accession numbers EU423905.1, EF489022.1, and EU423903.1.
In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yr46 gene. Each possibility represents a separate embodiment of the invention. In so embodiments, the artificial vector further comprises a Yr46 gene. In some embodiments, Yr46 is the Lr67 gene. In some embodiments, the Yr46 gene is operably linked to a plant promoter. In some embodiments, the artificial vector further comprises a nucleic acid sequence that encodes for a Yr46 protein. In some embodiments, Yr46 comprises or consists of the sequence
In some embodiments, the artificial vectors of the invention comprise at least one promoter for transcription in a plant cell. In some embodiments, the artificial vectors of the invention comprise at least one promoter for expression in a plant cell. In some embodiments, the plant is a grain/cereal plant. In some embodiments, the grain/cereal plant is wheat. In some embodiments, the grain plant is selected from barley, rye, triticale, oat, triticale, spelt and wheat. In some embodiments, the grain plant is selected from barley, rye, rice, maize, triticale, oat, triticale, spelt and wheat. In some embodiments, the at least one promoter is operably linked to an isolated DNA of the invention. In some embodiments, the at least one promoter is operably linked to a Yr5 gene. In some embodiments, the artificial vector comprises a DNA of the invention and the Yr5 gene. In some embodiments, the DNA of the invention and the Yr5 gene are operably linked to the same promoter. In some embodiments, the DNA of the invention and the Yr5 gene are operably linked to different promoters.
As used herein, the term “wheat” refers to a plant of the genus Triticum. Wheat can be used for the production of grain such as is used for bread, cereal or pasta for non-limiting examples. In some embodiments, wheat is bread wheat or duram wheat. In some embodiments, wheat comprises spelt. In some embodiments, wheat is Triticum turgidum.
By another aspect there is provided a transgenic plant cell, comprising an isolated DNA of the invention or artificial vector of the invention. As used herein, a “transgenic cell” refers to a cell that has undergone human manipulation on the genomic or gene level. In some embodiments, the transgenic cell has had exogenous DNA introduced into it. In some embodiments, a transgenic cell comprises a cell that has an artificial vector introduced into it. In some embodiments, a transgenic cell is a cell which has undergone genome mutation or modification. In some embodiments, a transgenic cell is a cell that has undergone CRISPR genome editing. In some embodiments, a transgenic cell is a cell that has undergone targeted mutation of at least one base pair of its genome. In some embodiments, the DNA or vector is stably integrated into the cell. In some embodiments, the transgenic cell expresses a DNA of the invention. In some embodiments, the transgenic cell expresses a vector of the invention. In some embodiments, the transgenic cell expresses a protein of the invention. In some embodiments, the transgenic cell, is a cell that comprises a wtk1 non-functional allele and/or pseudogene that has been mutated or modified into a functional WTK1 gene. In some embodiments, the wtk1 non-functional allele and/or pseudogene has been modified to comprise SEQ ID NO: 1. In some embodiments, the wtk1 non-functional allele and/or pseudogene has been modified to comprise SEQ ID NO: 3. In some embodiments, a wtk1 non-functional allele has been modified to encode a protein comprising the amino acid sequence provided in SEQ ID NO:2. In some embodiments, a wtk1 non-functional allele has been modified to encode a protein comprising an amino acid sequence with at least 99%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO:2, and which confers resistance to Pst. In some embodiments, CRISPR technology is used to modify a wtk1 non-functional allele.
In some embodiments, the plant is a grain/cereal plant. In some embodiments, the plant is any plant that without addition of the vectors or nucleic acids of the invention can be infected by Pst. In some embodiments, the plant is selected from barley, rye, triticale, oat, and wheat. In some embodiments, the plant is selected from barley, rye, triticale, oat, wheat, rice and maize. In some embodiments, the plant is wheat. In some embodiments, the transgenic plant cell is resistant to Pst. In some embodiments, the transgenic plant cell cannot be infected by Pst. In some embodiments, Pst does not grow on the transgenic plant cell. In some embodiments, Pst grows poorly on the transgenic plant cell. In some embodiments, Pst grows worse on the transgenic plant cell than on a plant cell that does not comprise a vector or nucleic acid molecule of the invention.
By another aspect there is provided a transgenic plant, or any portion, seed, tissue or organ thereof, comprising at least one transgenic plant cell of the invention. In some embodiments, the transgenic plant or portion thereof consists of transgenic plant cells of the invention. In some embodiments, the plant or portion thereof comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% transgenic cells of the invention. Each possibility represents a separate embodiment of the invention.
In some embodiments, the plant is a grain/cereal plant. In some embodiments, the plant is any plant that without addition of the vectors or nucleic acids of the invention can be infected by Pst. In some embodiments, the plant is selected from barley, rye, triticale, oat and wheat. In some embodiments, the plant is selected from barley, rye, triticale, oat, wheat, rice and maize. In some embodiments, the plant is wheat. In some embodiments, the transgenic plant is resistant to Pst. In some embodiments, the transgenic plant cannot be infected by Pst. In some embodiments, Pst does not grow on the transgenic plant. In some embodiments, Pst grows poorly on the transgenic plant. In some embodiments, Pst grows worse on the transgenic plant than on a plant that does not comprise a transgenic cell of the invention.
By another aspect, there is provided an isolated protein encoded by an isolated DNA of the invention. In some embodiments, the isolated protein comprises an amino acid sequence with at least 80% homology to SEQ ID NO:2, and wherein the isolate protein confers resistance to Pst. In some embodiments, the isolated protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments the isolated protein consists SEQ ID NO: 2. In some embodiments, the isolated protein is WTK1. In some embodiments, the isolated protein comprises or consists of an analog to WTK1 or SEQ ID NO: 2. In some embodiments, the isolated protein comprises or consists of a fragment to WTK1 or SEQ ID NO: 2. In some embodiments, the isolated protein comprises or consists of a derivative of WTK1 or SEQ ID NO: 2. In all such embodiments, it will be understood that the protein will retain the ability to confer resistance to Pst to a cell or plant to which it is introduced.
The term “derivative” as used herein, refers to any polypeptide that is based off the polypeptide of the invention and still confers resistance to Pst. A derivative is not merely a fragment of the polypeptide, nor does it have amino acids replaced or removed (an analog), rather it may have additional modification made to the polypeptide, such as post-translational modification. Further, a derivative may be a derivative of a fragment of the polypeptide of the invention, however, in such a case the fragment must comprise at least 100 consecutive amino acids of the polypeptide of the invention.
In some embodiments, the isolated protein of the invention comprises KinI and KinII. In some embodiments, KinI and KinII are separated by a spacer. In some embodiments, the spacer is at least 1, 3, 5, 10, 15, 20, or 25 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the isolated protein comprises a functional analog or derivative of KinI and/or KinII. It will be understood that this functionality is determined by the ability to confer resistance to Pst.
By another aspect, there is provided a nucleic acid molecule capable of binding differentially to a functional Wtk1 nucleic acid molecule and a non-functional wtk1 nucleic acid molecule, wherein a functional Wtk1 confers resistance to Stripe Rust (Pst). In some embodiments, the sequence of the functional Wtk1 is selected from SEQ ID NO: 1 and SEQ ID NO: 3. In some embodiments, the sequence of the functional Wtk1 comprises two kilobases upstream and/or downstream of the Wtk1 genomic DNA provided in SEQ ID NO: 3). In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.
As used herein, a “non-functional allele” and “wtk1” are used interchangeably and refer to an allele with high identity to Wtk1, but which lacks the ability to make a functional protein that provides resistance to Pst. In some embodiments, the non-functional allele comprises an insertion, deletion and/or mutation in the coding region. In some embodiments, the insertion, deletion and/or mutation results in a stop codon being introduced and low or no production of full-length protein. In some embodiments, a non-functional allele comprises a point-mutation in KinI and/or KinII. In some embodiments, a non-functional allele is a pseudogene of Wtk1
In some embodiments, the sequence of the non-functional wtk1 is from T. dicoccoides accession Zavitan. In some embodiments, the sequence of the non-functional wtk1 comprises
In some embodiments, the sequence of the non-functional wtk1 consists of SEQ ID NO: 111).
In some embodiments, the sequence of the non-functional wtk1 is from T. aestivum cultivar name (cv.) Chinese spring. In some embodiments, the sequence of the non-functional wtk1
In some embodiments, the sequence of the non-functional wtk1 consists of SEQ ID NO: 112).
In some embodiments, the sequence of the non-functional wtk1 is from T. durum cv. Kronos. In some embodiments, the sequence of the non-functional wtk1 comprises
In some embodiments, the sequence of the non-functional wtk1 consists of SEQ ID NO: 113).
In some embodiments, the sequence of the non-functional wtk1 is from T. aestivum cv. Cadenza_1b. In some embodiments, the sequence of the non-functional wtk1 comprises
In some embodiments, the sequence of the non-functional wtk1 consists of SEQ ID NO: 114).
In some embodiments, the sequence of the non-functional wtk1 is from T. aestivum cv. Claire. In some embodiments, the sequence of the non-functional wtk1 comprises
In some embodiments, the sequence of the non-functional wtk1 consists of SEQ ID NO: 115).
In some embodiments, the sequence of the non-functional wtk1 is from T. aestivum cv. Paragon. In some embodiments, the sequence of the non-functional wtk1 comprises
In some embodiments, the sequence of the non-functional wtk1 consists of SEQ ID NO: 116).
In some embodiments, the sequence of the non-functional wtk1 is from T. aestivum cv. Robigus. In some embodiments, the sequence of the non-functional wtk1 comprises
In some embodiments, the sequence of the non-functional wtk1 consists of SEQ ID NO: 117).
In some embodiments, the sequence of the non-functional wtk1 comprises any one of SEQ ID NO: 111-117. In some embodiments, the sequence of the non-functional wtk1 consists of any one of SEQ ID NO: 111-117. In some embodiments, the sequence of the non-functional wtk1 comprises the introns associated with any one of SEQ ID NOs: 111-117. In some embodiments, the sequence of the non-functional wtk1 comprises 2 kilobases upstream and/or downstream of any part of SEQ ID NOs: 111-117.
In some embodiments, the nucleic acid molecule capable of binding differentially is a primer. As used herein, the term “primer” includes an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3′ end along the template so that an extended duplex is formed. Primers within the scope of the present invention bind at the target sequence. A “primer” may be considered a short polynucleotide, generally with a free 3′-OH group that binds to a target or template potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target. Primers of the invention are comprised of nucleotides ranging from 8 to 30 nucleotides. In one aspect, the primer is at least 8 nucleotides, or alternatively at least 9 nucleotides, or alternatively at least 10 nucleotides, or alternatively at least 11 nucleotides, or alternatively at least 12 nucleotides, or alternatively at least 13 nucleotides, or alternatively at least 14 nucleotides, or alternatively at least 15 nucleotides, or alternatively at least 16 nucleotides, or alternatively at least 17 nucleotides, or alternatively, at least 18 nucleotides, or alternatively, at least 19 nucleotides, or alternatively, at least 20 nucleotides, or alternatively, at least 21 nucleotides, or alternatively, at least 22 nucleotides, or alternatively, at least 23 nucleotides, or alternatively, at least 24 nucleotides, or alternatively, at least 25 nucleotides, or alternatively, at least 26 nucleotides, or alternatively, at least 27 nucleotides, or alternatively, at least 28 nucleotides, or alternatively, at least 29 nucleotides, or alternatively, at least 30 nucleotides. In one embodiment, the primer is at most 40 nucleotides, or alternatively at most 50 nucleotides, or alternatively at most 75 nucleotides or alternatively at most 100 nucleotides.
As used herein, “differential binding” refers to the ability to bind to one molecule with greater affinity than another molecule. In some embodiments, differential binding is the ability to bind to one molecule and to not bind the other molecule at all. In some embodiments, differential binding is the ability to bind to one molecule and to negligibly bind the other molecule. In some embodiments, differential binding is at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% greater binding to one molecules as compared to the other. Each possibility represents a separate embodiment of the invention. In some embodiments, the molecule binds better to Wtk1. In some embodiments, the molecule binds better to wtk1. In some embodiments, the molecule binds with 100% complementarity to Wtk1, but only negligibly with wtk1. In some embodiments, the molecule binds with 100% complementarity to wtk1, but only negligibly with Wtk1. In some embodiments, the molecule binds with 100% complementarity to Wtk1 or wtk1, but not to both. In some embodiments, the molecule binds with 100% complementarity to Wtk1, and with less than 99%, 98%, 97%, or 96% complementarity to wtk1. Each possibility represents a separate embodiment of the invention. In some embodiments, the molecule binds with 100% complementarity to wtk1, and with less than 99%, 98%, 97%, or 96% complementarity to Wtk1. Each possibility represents a separate embodiment of the invention. In some embodiments, the molecule binds with 100% complementarity to Wtk1, and all nucleotides of the primer complement wtk1 except for the most 3′ nucleotide. In some embodiments, the molecule binds with 100% complementarity to wtk1, and all nucleotides of the primer complement Wtk1 except for the most 3′ nucleotide.
In some embodiments, the nucleic acid molecule with differential binding comprises at least one single nucleotide polymorphism (SNP) that differentiates between a functional Wtk1 nucleic acid molecule and a non-functional wtk1 nucleic acid molecule. SNPs are well known in the art, and are used for genotyping different alleles of a gene or genomic region. Any nucleotides which are different between the functional Wtk1 provided herein and a non-functional wtk1 are to be considered as SNPs that may be used for practicing the invention. Examples of such SNPs are provided in Table 15.
T. aestivum
T.
T.
T. durum
T.
Cadenza, Claire,
dicoccoides
dicoccoides
Svevo and
aestivum
Paragon and
Zavitan
Kronos
Robigus
T, A
T, G
T, A
T, —
T, G
The term “complementary” refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenosine. However, when a U is denoted in the context of the present invention, the ability to substitute a T is implied, unless otherwise stated. Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 90% complementarity.
As used herein, the term “confers resistance” refers to increasing the survival of a plant or cell when challenged with a pathogen i.e. Pst. In some embodiments, increasing survival is a decrease in the growth of the pathogen. In some embodiments, increasing survival is a decrease in the spread of the pathogen. In some embodiments, increasing survival comprises lack of infection by the pathogen. In some embodiments, resistance comprises not being able to be infected by the pathogen. In some embodiments resistance comprises increased survival as compared to survival without the vector, DNA, or protein of the invention. In some embodiments, the increase refers to at least 10% increase, 20% increase, 30% increase, 40% increase, 50% increase, 60% increase, 70% increase, 80% increase, 90% increase, or 100% increase in survival. Each possibility represents a separate embodiment of the invention. In some embodiments, the decrease refers to at least 10% decrease, 20% decrease, 30% decrease, 40% decrease, 50% decrease, 60% decrease, 70% decrease, 80% decrease, 90% decrease, or 100% decrease. Each possibility represents a separate embodiment of the invention.
In some embodiments, a molecule of the invention comprises the sequence provided in any one of SEQ ID NO: 69-70 and 103-110. In some embodiments, a molecule of the invention consists of the sequence provided in any one of SEQ ID NO: 69-70 and 103-110. In some embodiments, a molecule of the invention comprises the sequence provided in any one of SEQ ID NO: 61-110. In some embodiments, a molecule of the invention consists of the sequence provided in any one of SEQ ID NO: 61-110.
By another aspect, there is provided an isolated nucleic acid molecule comprising the sequence of any one of SEQ ID NOs: 61-110. In some embodiments, the nucleic acid molecule consists of a sequence provided in one of SEQ IDNOs: 61-110. By another aspect, there is provided an isolated nucleic acid molecule comprising the sequence of any one of SEQ ID NOs: 69-70 and 103-110. In some embodiments, the nucleic acid molecule consists of a sequence provided in one of SEQ ID 69-70 and 103-110.In some embodiments, the isolated nucleic acid molecule is a primer.
In some embodiments, the isolated molecules of the invention further comprise a tag. As used herein, a “tag” is any moiety or molecule that can be used to identify or isolate the molecule of the invention. In some embodiments, the tag is selected from a fluorescent tag, a chemiluminescent tag, a colored dye tag, a radioactive tag, a chemically modified nucleotide and a protein including an enzyme, a peptide or a ligand. In some embodiments, the fluorescent tag is a fluorescence resonance energy transfer (FRET) tag.
The tag may be identified by any means known to one skilled in the art, and may include, but is not limited to, microscopy, fluorescent sorting, binding to affinity substrates and high-performance liquid chromatography (HPLC). In some embodiments, PCR followed by measuring fluorescence is used to identify and quantify the tag. In some embodiments, FRET is used to identify and quantify the tag.
In some embodiments, the primers and nucleic acid molecules of the invention are for use in detecting a functional Wtk1 gene in a sample. In some embodiments, the primers and nucleic acid molecules of the invention are for use in determining the functionality of a Wtk1 gene in a sample.
By another aspect, there is provided a method of conferring resistance to Pst to a plant cell, the method comprising expressing in the cell at least one of an isolated DNA molecule of the invention, an artificial vector of the invention and a protein of the invention, thereby conferring resistance to Pst to a plant cell. By another aspect, there is provided a method of conferring resistance to Pst to a plant or cell thereof, the method comprising expressing in a cell of the plant at least one of an isolated DNA molecule of the invention, an artificial vector of the invention and a protein of the invention, thereby conferring resistance to Pst to a plant or cell thereof.
In some embodiments, the expressing is in at least 20, 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 99 or 100% of the cells of the plant. Each possibility represents a separate embodiment of the invention.
By another aspect, there is provided a method of conferring resistance to Pst to a plant cell, the method comprising converting at least one wtk1 non-functional allele of the cell into a functional Wtk1 gene, thereby conferring resistance to Pst to a plant cell.
In some embodiments, the plant cell is not resistant to Pst. In some embodiments, the plant cell does not comprise a functional Wtk1 gene. In some embodiments, the cell comprises a wtk1 non-functional allele. In some embodiments, the cell comprises a wtk1 pseudogene.
In some embodiments, said converting comprises CRISPR genome editing. In some embodiments, said converting comprises targeted mutation of said wtk1 non-functional allele and/or pseudogene. In some embodiments, the functional Wtk1 gene comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO:1. Each possibility represents a separate embodiment of the invention. In some embodiments, the functional Wtk1 gene comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO:3. Each possibility represents a separate embodiment of the invention. In some embodiments, the functional Wtk1 gene comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO: 119. Each possibility represents a separate embodiment of the invention. In some embodiments, the functional Wtk1 gene comprises a nucleic acid sequence that encodes a protein with at least 99%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO: 2 and confers resistance to Pst. Each possibility represents a separate embodiment of the invention.
By another aspect, there is provided a method of conferring resistance to a pathogen to a plant or cell thereof, the method comprising expressing in a cell of the plant at least one DNA sequence comprising a TKP-containing gene or at least one DNA sequence encoding a TKP-containing protein. In some embodiments, the TKP-containing gene is not a Yr gene. In some embodiments, the TKP-containing gene is not a wheat gene. In some embodiments, the TKP-containing gene is not a barley gene. In some embodiments, the TKP-containing gene is not RPG1. In some embodiments, the TKP-containing gene is not MLOC. In some embodiments, the TKP-containing gene is not a cereal plant gene. In some embodiments, the DNA sequence comprises a homolog or fragment of the TKP-containing gene that confers resistance In some embodiments, the TKP-containing gene is a T. aestivum gene. In some embodiments, the TKP-containing gene is a H. vulgare gene. In some embodiments, the TKP-containing gene is not a H. vulgare gene. In some embodiments, the TKP-containing gene is a S. cereale gene. In some embodiments, the TKP-containing gene is a O. sativa gene. In some embodiments, the TKP-containing gene is a Z. mays gene. In some embodiments, the TKP-containing gene is an A. thaliana gene. In some embodiments, the TKP-containing gene is a P. patens gene. In some embodiments, the TKP-containing gene is a S. tuberosum gene. In some embodiments, the TKP-containing gene is a B. napus gene. In some embodiments, the TKP-containing gene is a P. trichocarpa gene. In some embodiments, the TKP-containing gene is a S. bicolor gene. In some embodiments, the TKP-containing gene is selected from a wheat, barley, rye, rice, maize, thale cress, earth moss, potato, rapeseed, black cottonwood and sorghum gene. In some embodiments, the TKP-containing gene is selected from a wheat, rye, rice, maize, thale cress, earth moss, potato, rapeseed, black cottonwood and sorghum gene. In some embodiments, the TKP-containing gene is a wheat gene. In some embodiments, the TKP-containing gene is a rye gene. In some embodiments, the TKP-containing gene is a rice gene. In some embodiments, the TKP-containing gene is a maize or corn gene. In some embodiments, the TKP-containing gene is a thale cress gene. In some embodiments, the TKP-containing gene is a earth moss gene. In some embodiments, the TKP-containing gene is a potato gene. In some embodiments, the TKP-containing gene is a rapeseed gene. In some embodiments, the TKP-containing gene is a black cottonwood gene. In some embodiments, the TKP-containing gene is a sorghum gene.
By another aspect, there is provided a method for detecting a functional Wtk1 gene in a sample, comprising:
thereby, detecting a functional Wtk1 gene in a sample.
In some embodiments, the detecting comprises:
By another aspect, there is provided a method for determining functionality of a Wtk1/wtk1 gene in a sample, comprising:
Detecting a product of amplification is well known in the art and includes but is not limited to gel electrophoreses and column purification. In some embodiments, the one or more primers comprise a tag and the detecting a product of the amplifying comprises detecting the tag. In some embodiments, the tag is a fluorescent tag. In some embodiments, the tag is a FRET tag. Fluorescently-tagged PCR is well known in the art, and detection of the amplification product may be performed with any fluorometer.
In some embodiments, a functional Wtk1 gene comprises the nucleic acid sequence provided in SEQ ID NO: 1. In some embodiments, functional Wtk1 gene comprises the nucleic acid sequence provided in SEQ ID NO: 3. In some embodiments, functional Wtk1 gene comprises the nucleic acid sequence provided in SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the functional Wtk1 gene encodes a protein with two functional kinase domains. In some embodiments, the kinase domains comprise the sequences provided in SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, a functional Wtk1 gene encodes a protein that confers resistance to Pst.
In some embodiments, the sample is from a plant. In some embodiments, the plant is a cereal. In some embodiments, the cereal is selected from wheat, barley, oats, triticale and rye. In some embodiments, the cereal is selected from wheat, barley, oats, triticale, rye, rice and maize. In some embodiments, the cereal is wheat. In some embodiments, the plant is any plant that can be infected by Pst.
In some embodiments, the sample is from a leaf of the plant. In some embodiments, the sample is from a mature plant. In some embodiments, the sample is from any one of cultivated plant germplasm, pre-breeding materials, and elite plant cultivars. As used herein, “germplasm” refers to any living tissue from which a new plant can be grown. In some embodiments, the germplasm is a seed. In some embodiments, the germplasm is any one of a seed, a leaf, stem, tissue culture cells, embryoids and pollen. In some embodiments, the germplasm comprises only a few cells. In some embodiments, the germplasm comprises enough material to perform PCR. As used herein, “pre-breeding materials” refers to materials that are not generally directly be used for breeding, but which contain genetic information that can be transferred to breeding materials. As used herein, “cultivars” refers to a plant or group of plants selected for desirable characteristics and maintained by propagation. It will be understood by one skilled in the art, that it is advantageous to those growing cereal plants, to integrate a functional Wtk1 gene into the genomes of their crops. Further, it will be advantageous to integrate it into all of the crop and not just a portion. As such, the grower will need to confirm the presence of the functional gene, potentially at every stage of the transfer of the gene to the crop. The molecules and methods of the invention can be used for this purpose, and thus the methods can be performed at any step of the process of integrating Wtk1 into their crops, and with any material that might be used in this process.
In some embodiments, the provided nucleic acids comprise at least one of genomic DNA, RNA and cDNA reverse-transcribed from RNA from the sample. In some embodiments, the hybridizing comprises at least one of, PCR, southern blotting and northern blotting. One skilled in the art will appreciate that the method of hybridization will be selected to match the source of the provided nucleic acids. As non-limiting examples, PCR may be selected when the nucleic acids are cDNA, southern blotting may be selected when the nucleic acids are genomic DNA and northern blotting may be selected when the nucleic acids are RNA. In some embodiments, the PCR is any one of RT-PCR, qPCR, real-time PCR, or conventional end-point PCR. In some embodiments, detecting the hybridizing comprises detection of a PCR product. In some embodiments, the detecting comprises gel electrophoreses. In some embodiments, the detecting comprises sequencing, deep sequencing or next-generation sequencing.
In some embodiments, the primers are Kompetative Allele Specific PCR (KASP) primers. KASP is a well-known fluorescently tagged PCR amplification assay. It requires three primers, two differential primers, one each for two genes (or alleles) and a third primer that is common to both genes/alleles. In some embodiments, the KASP assay can be multiplexed, with two sets of three primers used at the same time for amplification. In some embodiments, two amplifications are performed separately. In some embodiments, a primer comprising the sequence of SEQ ID NO: 105, a primer comprising the sequence of SEQ ID NO: 106, and a primer comprising the sequence of SEQ ID NO: 107 are used in the method. In some embodiments, a primer comprising the sequence of SEQ ID NO: 108, a primer comprising the sequence of SEQ ID NO: 109, and a primer comprising the sequence of SEQ ID NO: 110 are used in the method. In some embodiments, a primer comprising the sequence of SEQ ID NO: 105, a primer comprising the sequence of SEQ ID NO: 106, a primer comprising the sequence of SEQ ID NO: 107, a primer comprising the sequence of SEQ ID NO: 108, a primer comprising the sequence of SEQ ID NO: 109, and a primer comprising the sequence of SEQ ID NO: 110 are used in the method.
By another aspect, there is provided a method of designing a differential primer for differentiating between Wtk1 and wtk1, the method comprising selecting a primer with 100% complementarity to either Wtk1 or wtk1, and at least one nucleotide that is not complementary to the nucleotide at the same position in the gene for which there is not 100% complementarity. A person skilled in the art will understand that the mores 3′ nucleotide of a primer are the most crucial for binding, and thus a single nucleotide polymorphism (SNP) between Wtk1 and wtk1 positioned at the 3′ end will confer differential binding. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are not complementary to the gene for which the primer does not have 100% complementarity. Each possibility represents a separate embodiment of the invention. In some embodiments, the most 3′ nucleotide is not complementary. In some embodiments, at least one of the three most 3′ nucleotides are not complementary.
By another aspect there is provided a kit comprising at least 2 primers selected from a primer comprising the sequence of SEQ ID NO: 105, a primer comprising the sequence of SEQ ID NO: 106, and a primer comprising the sequence of SEQ ID NO: 107, and/or at least 2 primers selected from a primer comprising the sequence of SEQ ID NO: 108, with a primer comprising the sequence of SEQ ID NO: 109, and a primer comprising the sequence of SEQ ID NO: 110.
By another aspect there is provided a kit comprising at least 2 primers, wherein the primers comprise a sequence selected from SEQ ID NOs: 69-70 and 103-110.
In some embodiments, the kit comprises a primer comprising the sequence of SEQ ID NO: 105, and a primer comprising the sequence of SEQ ID NO: 107 and optionally a primer comprising the sequence of SEQ ID NO: 106. In some embodiments, the kit comprises a primer comprising the sequence of SEQ ID NO: 108, and a primer comprising the sequence of SEQ ID NO: 110 and optionally a primer comprising the sequence of SEQ ID NO: 109.
In some embodiments, at least one primer of the kit comprises a tag. In some embodiments, the differential primers of the kit comprise a tag. In some embodiments, the tag is selected from a fluorescent tag, a chemiluminescent tag, a colored dye tag, a radioactive tag, and a protein including an enzyme, a peptide or a ligand. In some embodiments, the tag is a FRET tag. In some embodiments, the primers are KASP primers.
In some embodiments, the kits of the invention are for use in determining the functionality of a Wtk1/wtk1 gene in a sample. In some embodiments, the kits of the invention are for use in detecting a functional Wtk1 gene or a non-functional wtk1 gene in a sample.
As used herein, the term “about” when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+−100 nm.
It is noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
In those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994 Mishell and Shiigi (eds)), “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.
A large F2 mapping population was developed by crossing the susceptible durum wheat (Triticum turgidum L ssp. durum) accession D447 (LD393/2*Langdon ND58-322) as the female parent with the resistant either BC3F9(B9) or BC3F10 (B10) introgression line, which carry the Yr15 gene within a 1BS chromosome segment introgressed from wild emmer wheat [T. turgidum L ssp. dicoccoides (DIC)], accession G25 (G25), as the male parent. A collection of Yr15 introgression lines (IL), representing a wide range of durum wheat (tetraploid) and common wheat (hexaploid) varieties, was used in the current study (Table 12). In addition, the following Yr5/Yr15 ILs, which were developed by J. Dubcovsky and represent an example for pyramiding of resistance genes, were challenged with several stripe rust [Puccinia striiformis f sp. tritici (Pst)] races (pedigrees in parentheses): YecoraRojoYr5Yr15 (Ciano 67//Sonora 64/Klein Rendidor/3/II-8156=1123584), PatwinYr5Yr15 (Madsen/2*Express), SummitYr5Yr15 (Express//Tadorna/PB775), DirkwinYr5Yr15 (Twin/Triple Dirk). The Yr15 gene knockout mutagenized lines were developed by ethyl methane sulphonate (EMS) treatment of the tetraploid IL B9, and the hexaploid ILs Avocet+Yr15, Excalibur+Yr15, and Suncea+Yr15. The hexaploid common wheat variety ‘Fielder’ and the tetraploid durum wheat ‘Kronos’, which are susceptible to Israeli Pst isolate #5006 (race 38E134; 16) and U.S. Pst race PST-130 (17) were used for the transgenic complementation experiment. The Triticum accessions evaluated for the distribution of Yr15 alleles were kindly provided by the Centre for Genetic Resources (CNG; Wageningen University & Research, Wageningen, The Netherlands), and by the USDA National Small Grain Collection (NSGC; USDA, Aberdeen, Id., USA). The DIC accessions that were collected from a broad range of natural habitats in the Fertile Crescent are maintained at the Wild Cereal Gene Bank of the Institute of Evolution, University of Haifa (Haifa, Israel).
Phenotyping of recombinant, mutant and transgenic lines under growth chamber conditions was carried out in Haifa University and UC Davis according to the following protocol: urediniospores used for inoculation of leaf tissue were first suspended in Soltrol® 170 light oil (Chevron Phillips Chemical Company, The Woodlands, Tex.) and then sprayed using a TLC spray tube (Kontex, Blomberg, Germany). Inoculated plants were first placed in a dew chamber (100% humidity) at 10° C. for 16 h in the dark followed by 8 h of light. The plants were then transferred to a growth chamber (70% humidity) under the following day/night regime: 16 h at 15° C. with a light intensity of 150 μmol m−2 s−1 followed by 8 h at 10° C. in darkness. Plants were inoculated either at the two- to four-leaf stage (“seedling inoculation”) or at the stem elongation stage (“adult-plant inoculation”). The rust severity was evaluated and characterized 14 to 18 days after inoculation using a 0 to 9 scale of infection type (IT).
In the Global Rust Reference Centre (GRRC; Aarhus University, Denmark) Yr15 and Yr5 introgression lines were phenotyped for their response to the Pst isolates DK92/02 and AU85569 (
Microscopy of Pst-Wheat Interactions within Infected Leaf Tissues
Fluorescence microscopy of P. striiformis structures was performed. Wheat germ agglutinin (WGA; a lectin that binds specifically to β (1→4)-N-acetyl-D-glucosamine, i.e., chitin) conjugated with a fluorescent dye was used to visualize the intercellular growth and pustule formation on infected leaves. Leaf segments (2nd leaf, 10 cm long) from the near isogenic lines (NILs), Avocet S and Avocet+Yr15, each of which had been inoculated with urediniospores of Pst race #5006, were sampled from 1 until 11 day post inoculation (dpi) (10:00 to 10:30 am, every two days), when visible sporulation developed on the susceptible plants. The progress in visible response to Pst inoculation 1 to 14 dpi in the susceptible and the resistant lines is presented in
The sampled leaf segments were cleared with KOH at 37° C. for 24 h and washed twice with Tris-HCl (pH 7.5) for neutralization. Then, a solution of WGA conjugated to fluorophore Alexa 488 (L4895-2MG; Sigma-Aldrich) was placed on the leaf surfaces and stained for 24 h at 4° C. After washing with ddH2O, the stained leaf tissues were gently placed on microscope slides, immersed with antifade mounting medium for fluorescence (Vectashield, Vector Laboratories), covered with a cover glass, sealed with rubber cement, and stored at 4° C. in the dark. Fluorescent microscopy was carried out on an inverted fluorescent microscope, Leica DMi8 (Leica Microsystems, Wetzlar, Germany) fitted with a filter cube for the FITC excitation range (EX: 460-500, DC: 505, EM: 512-542) and a FLUO regime to observe the WGA-stained fungal structures. The resultant micrographs are presented in
Relative Quantification of Fungal Biomass within Infected Leaf Tissues
After inoculation with Pst isolate #5006 leaf segments (middle, 2nd leaf) of the resistant Avocet+Yr15 and susceptible Avocet S NILs were cut every two days (1-14 dpi), and 10 biological replicates collected for each line at each time point. Quantification of fungal biomass by chitin measurement was conducted. Leaf segments were weighed and autoclaved in KOH solution, then washed with Tris-HCl (pH 7.0) to neutralize pH. Leaf tissues were then transferred to microcentrifuge tubes that contained 1 ml of Tris-HCl solution for each 200 mg of fresh leaf tissue, according to weights determined at harvest time. Leaf samples were homogenized in a TissueLyser II (Qiagen) and an aliquot of each sample suspension was transferred to a microcentrifuge tube containing WGA conjugated to fluorophore Alexa 488 (L4895-2MG; Sigma-Aldrich) dissolved in water. Three technical replicates were made for each tissue sample. Samples were mixed by repetitive pipetting and incubated for 60 min at room temperature. After staining, samples were centrifuged at 600×g for 3 min. The supernatant was removed by pipetting and the pellet washed three times and resuspended in Tris-HCl, and then transferred into black, 96-well microtiter fluorometric trays. Fluorescence was measured on a SpectraMax M2e Microplate Reader (Molecular Devices, Sunnyvale, Calif., USA) with five seconds of shaking before first read, 485-nm excitation and 535-nm emission wavelengths, a 1.0 s measurement time, and a cross pattern of well scanning yielding an average measurement per well. Statistical analyses of parameters associated with the accumulation of fungal biomass were made by the maximal likelihood estimation (MLE) method (SPSS) following the logistic growth model. The null hypothesis, for: no growth was tested by the likelihood ratio test. The results are presented in
A primary genetic map of Yr15 was developed using simple sequence repeat (SSR) markers that had been assigned to chromosome arm 1BS. These SSR markers, suitable for marker-assisted selection (MAS) of Yr15, were used in the current study for screening a large F2 mapping population for lines containing recombinations between the closest flanking SSR markers, enabling assignment of Yr15 to deletion bin Sat0.31 of chromosome arm 1BS. Thereafter, a dominant marker and two cleaved amplified polymorphic sequence (CAPS) markers were developed as described by Raats et al., based on expressed sequence tags (ESTs) assigned by Peng et al. to 1BS deletion bin Sat0.31 (Table 1). These markers were tested on the T. aestivum cv. Chinese Spring (CS) deletion lines 1AS-1, 1BS-4, and 1BS-9, which were produced by Endo and Gill. B-genome-specific primers were used to amplify marker alleles of the D447, B9, and B10 lines in order to mine polymorphisms, which were exploited for the development of informative PCR markers. The genetic map of the Sat0.31 region that contains Yr15 was further refined by the GenomeZipper approach using synteny with the orthologous regions of Brachypodium distachyon, Oryza sativa, Sorghum bicolor (Table 3). With this approach, eight markers were developed and the Yr15 region thereby narrowed down to a 0.3 cM interval between the nearest Yr15 flanking markers, uhw264 and uhw259 (
For development of Kompetitive Allele Specific PCR (KASP) markers, B9 and D447 parental lines were genotyped using the 15K wheat single-nucleotide polymorphism (SNP) array (Trait Genetics GmbH, Gatersleben, Germany). Polymorphic markers, residing on chromosome arm 1BS between the SSR markers barc8 and gwm273 that flank Yr15, were identified from their location on the consensus tetraploid wheat genetic map. Sequences of two SNP markers, RAC875_c826_839 and BS00022902_51, were converted to KASP markers using the Polymarker pipeline (Table 2).
A total of 8573 F2 plants from the D447×B9 or D447×B10 crosses were screened for recombination events in the Yr15 gene region. Three marker sets flanking Yr15 region were used for screening: (i) the SSR markers wmc406 and gwm273, flanking a 13.3 cM interval; (ii) the KAPS markers RAC875_c826_839 and BS00022902_51, flanking a 1.3 cM interval (Fig. S6); (iii) the length polymorphism marker uhw264 and the CAPS marker uhw259, flanking a 0.3 cM interval (
Markers uhw264 and uhw259, which flank Yr15, were used to screen a gridded CS BAC library for clones corresponding to the Yr15 region. Three positive BAC clones (TaaCsp364011, TaaCsp1023G2 and TaaCsp1158K20) were picked with marker uhw264 and two (TaaCsp729H14 and TaaCsp814G12) with uhw259 (
The Yr15 donor line, G25, was used for construction of a pooled BAC library. High molecular weight genomic DNA was partially digested with Hind III to obtain fragment sizes in the range of 100-250 kb, which were ligated into a pINDIGO vector (Caltech, Pasadena, Calif., USA), and transformed into Escherichia coli cells. After growing individual E. coli colonies on agar plates, 443,880 transformed colonies were collected into 150 pools with an average of 2959 colonies per pool. The genome coverage of the G25 BAC library was calculated as 4.5× (average clone size 120 kb). From initial screening of the library with marker uhw280, BAC clone G25-64 was isolated and sequenced. Based on these sequences proximal markers uhw288, uhw289, uhw287, uhw285, uhw286, and uhw281 were developed (Table 4). Further screening of G25 BAC library with the closest distal markers uhw297 and uhw292, the co-segregating marker uhw296, and the proximal marker uhw273, yielded six BAC clones (G25-33, G25-86, G25-40, G25-141, G25-35 and G25-25;
As a first step prior to gene annotation, repetitive elements within the G25 BAC sequences were masked using the Triticeae Repeat Sequence Database (TREP; http://botserv2.uzh.ch/kelldata/trep-db/index.html). Then, the non-repetitive sequences were analyzed for genes by BLASTN searches against the TIGR Wheat Genome Database (http://tigrblast.tigr.org/euk-blast/index.cgi?project=tael), using the gene prediction software Genscan (http://genes.mit.edu/GENSCAN.html) and FGENESH (http://www.softberry.com/berry.phtml).
A blast search of sequences of the G25ctg1 and G25ctg2 contigs against the high confidence (HC) gene models of 1BS pseudomolecule of Zavitan revealed the presence of three putative HC genes that reside between the markers closest to Yr15. However, only one of them contained predicted protein domains that have been previously associated with plant responses to pathogens. This candidate gene, which has two different kinase-like domains arranged in tandem was designated as WHEAT TANDEM KINASE 1 (Wtk1) and was selected first for further validation (
Isolation and Sequencing of the Full-Length WTK1 cDNA
Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, Calif., USA) and poly (A)+RNA was purified from the total RNA with a Qligotex mRNA Midi Kit (Qiagen). First-strand cDNA was synthesized with Superscript II (Invitrogen) using primer E1820 (Table 11). Nested PCR was carried out first with E1820 and the Yr15 5′UTR primer Y15F0 and then with E2146 (matching part of E1820, Table 11) and the 5′UTR primer Y15F2. The PCR products were purified, cloned and sequenced.
To determine the start site of the WTK1 transcript, we used 5′ RACE. In brief, the poly (A)+RNA was treated with tobacco acid pyrophosphatase (TAP, Epicentre, USA), then purified with an RNeasy Plant Mini Kit (Qiagen). An adapter (RNAoligo, Table 12) was ligated to the 5′ end of the RNA and reverse transcription carried out as above, priming with E1820. Amplification of the 5′ end was carried out by two rounds of PCR reactions, using primer 5′RACE (Table 11) and sequentially two gene-specific primers, Y15R2 and Y15R1 (Table 11).
EMS treatment was conducted. Seeds of the Yr15 tetraploid (B9) and hexaploid (Avocet+Yr15, Excalibur+Yr15, and Suncea+Yr15) introgression lines were treated with 0.4-0.75% EMS solution for 16-18 h at room temperature. Prior to sowing, the seeds were washed three times with 10% sodium thiosulfate and then twice in water (30 min each time), covered with Whatman paper and air-dried at 4° C.
EMS-treated M1 plants were grown in the experimental fields of the Institute of Evolution, University of Haifa, Israel or at the Plant Breeding Institute, University of Sydney, Cobbitty, Australia. Their spikes were covered with envelopes to avoid cross-pollination. A total of 1002 hexaploid and 2112 tetraploid M2 families (10-20 seeds per family) were first screened for resistance to stripe rust under field conditions in Israel (with Pst isolate #5006) or in Australia (with Pst isolate 110 E143A+). All M3 seedlings obtained from susceptible M2 plants were inoculated in a growth chamber with Pst isolate #5006 to confirm homozygosity of the recessive mutations. In total, 10 mutagenized lines were obtained at the M2 generation and validated at M3, as shown in
The susceptible plants from the EMS-treated M2 families were screened for mutations within the coding sequence of the Yr15 candidate gene Wtk1, using three gene-specific primers (Table 11). Amplified products were sequenced and compared for nucleotide variations by multiple sequence alignment. Furthermore, two overlapping primer pairs (WTK1_L2F and WTK_RE6, Table 11) designed along the coding sequence of the Wtk1 were used to amplify the full-length cDNA from the susceptible M2 plants and PCR products were sequenced to confirm the detected point mutations. The Wtk1 mutations were ranked using the program Sorting Intolerant From Tolerant (SIFT) to predict the effects of non-synonymous mutations on protein function (Table 7). The independence of 10 mutagenized lines was confirmed, as detailed in Table 7.
We crossed two of the EMS-mutagenized lines of common wheat (EMS4 and EMS6, Table 7) with the resistant wild-type parental line Avocet+Yr15 to produce segregating F2 families. The response of each F2 population to stripe rust was assessed at the seedling stage, and a Chi-squared test used to evaluate the goodness of fit for the observed and expected ratios in that population (Table 8). DNA was isolated from 40 F2 plants and sequence analysis was used to confirm co-segregation of homozygosity for the mutations in Wtk1 with the loss of resistance to Pst.
Agrobacterium tumefaciens-mediated transformation of susceptible durum and common wheat varieties, respectively Kronos and Fielder, served to further verify WTK1 function by complementation.
Phusion® High-Fidelity DNA Polymerase (New England BioLabs, USA) was used to amplify the WTK1 genomic region from G25 BAC clone G25-141 (
In total, 15 independent T0 plants in Kronos (Tyr15-K1 to Tyr15-K15) and seven T0 plants in Fielder (Tyr15-F1 to Tyr15-F7) were obtained. Three primer pairs (HpyF1/R1, Yr15TestF1/R1 and Y15K1_F2/Yr15P2; Table 11) were used to validate the presence of WTK1 in the transgenic plants. In addition, we extracted mRNA from all T0 plants and estimated the transcript levels of the three Wtk1 isoforms (Main isoform, Isoform2 and Isoform3) by qRT-PCR using Ubiquitin as the endogenous control. The transcript levels of Wtk1 in all T0 events in Kronos and Fielder and in non-transgenic controls are shown in
We germinated 10-25 T1 seeds from each transgenic event that expressed WTK1 and inoculated the plants with Pst race PST-130, which is virulent on Kronos and Fielder. All 12 positive T1 families in Kronos and the five positive T1 families in Fielder showed resistance to this Pst race. We genotyped all tested T1 plants and confirmed co-segregation of the resistance with the presence of the transgene. Five representative susceptible T1 plants which did not carry the transgene and five resistant T1 plants with the transgene are presented in
Furthermore, 5-10 independent plants each of the T2 families from transgenic events Tyr15-F1, Tyr15-F4, Tyr15-F5, Tyr15-F6, Tyr15-K7, Tyr15-K8, Tyr15-K10O, Tyr15-K12, and Tyr15-K15 were also tested with Pst isolate #5006, which is likewise virulent on Kronos and Fielder. All five T2 positive families in Kronos and the four positive T2 families in Fielder showed the resistance phenotype (
Gene Expression Analysis by the Quantitative Reverse Transcription PCR (qRT-PCR)
Expression analysis was conducted. Total RNA was isolated from various plant tissues (leaves, roots, and stems) using an RNeasy Plant Mini Kit (Qiagen, USA). First-strand cDNA was generated using the qScript™ Flex cDNA Synthesis Kit (Quanta Biosciences, USA). A Wtk1 expression survey in leaves, roots, and stems was conducted by RT-PCR with Ubiquitin as the endogenous control. The qRT-PCR was performed on a StepOne Plus Real-Time PCR system (Applied Biosystems, USA) using the following program: 95° C. for 20 s; 40 cycles of 95° C. for 3 s, and 60° C. for 30 s. The qRT-PCR reaction mixture contained the following components in a total volume of 10 μl: 5 μl Fast SYBR green master mix (Applied Biosystems); 2.5 μl diluted cDNA; 300 nM of each primer. The efficiency of each pair of primers was calculated using four 5-fold dilutions (1:1, 1:5, 1:25, and 1:125) in triplicates. Amplification efficiencies were higher than 95%. Transcript levels are expressed as linearized fold-Ubiquitin levels calculated by the formula 2 (Ubiquitin CT−Target CT)±standard error of the mean (SEM).
Alternative Splicing of WTK1 mRNA
Sequencing of 48 B9 cDNA clones, which were amplified with a poly(T) primer and Wtk1-specific primers, or Wtk1-specific primers alone revealed three alternative splicing variants (Main isoform, Isoform2, Isoform3,
To determine the relative transcript levels of these three splicing variants, we designed WTK1 isoform-specific PCR primers for each one of them (Table 11). These primers served to amplify the three variants from leaves of B9, which were collected after the following treatments: (i) before inoculation (0 h), as the control; (ii) after inoculation with spores of Pst isolate #5006 suspended in Soltrol® 170 light oil; (iii) a mock control, comprising plants sprayed with Soltrol® 170 light oil without Pst spores. Samples of treatments (ii) and (iii) were collected at 1, 3, 5, 7, and 9 days post-inoculation (dpi), with 6 biological replicates for each treatment at each time point. All data were subjected to statistical analysis using the general linear model with SPSS software. The expression results are presented in
Barley protoplasts were isolated from Hordeum vulgare cv. Bomi plants. Protoplasts (8×105) were electroporated with 20-40 μg DNA at 300 V/cm essentially as is known in the art, diluted into Gamborg's B-5 Basal Medium with minimal organics that contained 10% glucose, and then transferred into a glass-bottomed 35 mm microwell dish (MatTek Corporation, USA) for microscopy observations.
Electroporated protoplasts were cultured overnight at 22° C. before imaging under an inverted confocal laser scanning microscope (Leica TCS SP5 II, Leica Microsystems) with a 63×water immersion objective. The following light ranges were used: GFP (ex 488 nm, em 500-543 nm); DAPI (ex 405 nm, em 430-550 nm); chlorophyll autofluorescence (ex 488 nm, em 673-725 nm). The results are presented in
A WTK1 cDNA clone was prepared as described in Section 8 (Isolation and sequencing of full-length WTK1 cDNA). All expression vectors were created with a Multisite Gateway Three-Fragment Vector Construction Kit (Invitrogen). The 5′ entry clone was prepared by the BP reaction between pDONRP4-P1R and a 35S promoter fragment amplified from pBI221 (GenBank accession AF502128.1) using primers attB4F35S and attB1R35S (Table 11). The entry clone for the full-length WTK1 was created by carrying out the BP reaction between pDONR221 and the PCR product amplified from the WTK1 cDNA clone with primers attBlFKinase and attB2RYr15 (Table 12). The entry clone for the N-terminal and C-terminal kinase-like domains were similarly prepared using primers attB1FKinase and attB2Rkinase, and attB1Freg and attB2RYr15 (Table 11), respectively. The 3′-entry clone was prepared by carrying out the BP reaction with pDONRP2RP3 and a GFPnos fragment amplified from pVEC8_GFP (GenBank: FJ949107.1) by PCR using primers attB2FGFPnos and attB3RGFPnos (Table 11). The final clones used for transient transformation and expression in protoplasts were created by the LR reaction between the respective entry clones and the destination vector pDESTR4-R3 according to the instructions of the Gateway kit.
A BLASTP search of the NCBI non-redundant protein database was used to assign WTK1 kinase-like domains to specific kinase superfamily and to search for sequences of proteins similar to WTK1.
Multiple alignment of WTK1 kinase domains with 23 different plant kinase domains was performed using Clustal Omega with default parameters. In this analysis, we included the four closest kinases identified by BLASTP (WKS1, PTO and two WAK5), two kinase domains of RPG1, two kinase domains of H. vulgare MLOC_38442.1 and 15 kinase domains from known and putative plant pattern recognition receptors (PRRs), of which 14 are non-RD kinases and one is an RD-kinase (
The following sequence databases were used for searching and extracting WTK1 protein sequences, used for phylogenetic analysis:
a) Genome assembly of wild emmer wheat (Zavitan pseudomolecules);
b) Genome assembly of common wheat (CS pseudomolecules);
c) Genome assembly of barley H. vulgare (Morex pseudomolecules);
d) Genome assembly of A. speltoides (accession #29; https://wheat-urgi.versailles.inra.fr/Seq-Repository/Assemblies);
e) Genome assembly of A. tauschii;
f) Genome assembly of T. urartu;
g) Genome assembly of rye S. cereale;
h) The public resource eggNOG version 4.5 was searched for WTK1 Orthologous Group (OG) of proteins at different taxonomic levels. The resulted group of WTK1 orthologs, designated as ENOG4115QHQ, included 11 protein sequences belonging to nine different species. Two of these proteins were used for further phylogenetic analysis (B. distachyon BRADI2G38370.1 and O. sativa LOC_Os01g20880.1).
In total, we have selected for analysis 21 protein sequences; all sequence positions that contained gaps and missing data, as compared with WTK1 from G25 1BS, were eliminated from analysis. Therefore, the analysis was conducted using 263 amino acid residues out of 665 of the full length of WTK1 from G25 1BS. The evolutionary history of WTK1 was inferred using the Neighbor-Joining algorithm based on evolutionary distances computed using the Poisson correction method. The quality of the derived phylogeny of WTK1 was assayed using bootstrap test with 10,000 replicates. The analysis was performed with MEGA7 software. The obtained results are presented in
DIC accessions (Table 9) were obtained from the National Small Grains collection (NSGC; USDA, Aberdeen, Id., USA; PI numbers), the Wild Cereal Gene Bank (WCGB; Institute of Evolution, University of Haifa, Haifa, Israel; TD numbers), the International Center for Agricultural Research in Dry Areas (ICARDA; Rabat, Morocco; IG numbers), the Center for Genetic Resources (CGN; Wageningen University & Research, Wageningen, The Netherlands; CGN numbers). Germplasm samples of other Triticeae species (Table 10) were provided by CGN and NSGC. Durum and common wheat accessions (Table 12) were obtained either from Salamini, the University of California (Davis), or the University of Haifa collections of Yr15 introgression lines.
A set of four gene-specific, diagnostic markers was used to test the distribution of WTK1, two for kinase-like domain I (KinI) and two for kinase-like domain II (KinII) (Table 11). Dominant PCR markers were designed to identify the presence of the Wtk1 functional allele, amplifying a 1 kb PCR product for KinI and 2 kb for KinII.
Co-dominant KASP markers that can differentiate between the functional Wtk1 and the non-functional wtk1 alleles (Table 9) were designed using the sequence of Wtk1 from G25 and those of wtk1 from CS and Zavitan. Alignment of all WTK1 sequences was made with the BioEdit software. The positions of the SNPs in the coding regions of KinI and KinII that differentiate between Wtk1 and wtk1 sequences were determined and served in the design of the forward A and B KASP primers. The reverse common primer (C) was designed at the NCBI/Primer-BLAST website (https://www.ncbi.nlm.nih.gov).
A BLASTP search of the NCBI non-redundant protein database revealed that both WTK1 kinase-like domains belong to the superfamily of protein kinase catalytic domains. KinI, which is derived from the first exon of WTK1 (residues 42-310), contains all 11 conserved subdomains of a protein kinase. The nearly invariant residues of WTK1 KinI subdomain I (Gly-X-Gly-X-X-Gly) and subdomain VIII (Ala, Phe and Gly, Fig. S14) are all conserved; nevertheless, in subdomain VI, KinI has Gly residue instead of Arg adjacent to Asp. Thus, it is classified as a non-RD kinase (Fig. S14). Moreover, the conserved residues found in KinI subdomains VI and VIII classify it as a protein-serine/threonine kinase (
Structure of Wtk1 from CS and Zavitan
A search for WTK1 sequences in the chromosome arm 1BS of CS and Zavitan whole genome assemblies revealed the presence of non-functional alleles, designated as wtk1, in both of these susceptible lines. These alleles differ from the functional Wtk1 allele of G25 by large insertions in intron 1 and 3 for both CS and Zavitan, stop codon in exon 4 in Zavitan and indel in exon 4 for CS. A search in two databases of transposable elements (TE)-mipsREdat_9.3p Poaceae_TEs and trep-db_complete_Rel-16—identified the presence of three TEs: (i) RLX_Taes_Veju (intron 1 of CS and Zavitan); (ii) DTH_158357|trep3042|DNA/Harbinger (intron 3 of CS and Zavitan); (iii) DTM_Hvul_Spring_TREP1674-1 Hordeum vulgare; DNA-transposon, TIR, Mutator—MITE (intron 3 of CS). These results are presented in
Previously, Yr15 was mapped on the short arm of chromosome 1B. A recombinant inbred family segregating for Yr15 was generated by crossing resistant introgression durum lines (B9, B10), which carry Yr15 from DIC G25, with the susceptible recurrent line D447. 8573 F2 plants and 12 DNA markers derived from the collinear region in Brachypodium, Oryza, and Sorghum (Tables 1, 2, and 3) were used to map Yr15 0.17 cM distal to uhw259 and 0.13 cM proximal to uhw264 (
aMarker order from distal to proximal relative to Yr15, is described in FIG. 2.
B. distachyon, O. sativa, and S. bicolor.
A pooled BAC library, constructed for Yr15 donor G25, was screened with closely linked markers Xuhw292, Xuhw297, Xuhw296, and Xuhw273. The sequencing of the six BACs identified with these markers yielded two non-overlapping contigs (G25ctg1—286,738 bp and G25ctg2—131,485 bp) (
To validate the function of Wtk1, ethylmethane sulfonate (EMS) was used to mutagenize a set of Yr15 ILs and identified two out of 2,112 tetraploid and eight out of 1,002 hexaploid M2 families, that segregated for resistance to Pst (
1The first letter indicates the wild-type nucleotide, the number indicates its position relative to the ATG start codon, and the last letter shows the mutant nucleotide.
2The first letter indicates the wild-type amino acid, the number indicates its position relative to the start methionine, and the last letter shows the mutated amino acid.
3Rust severity was evaluated and characterized 14-18 days after inoculation with Pst isolate #5006 using a 0-9 scale of infection type (IT): 0-3 is considered resistant (R), 4-6 moderately resistant, and 7-9 susceptible (S). The complete WTK1 coding regions of the above ten mutagenized lines were sequenced and no additional mutations were detected.
4The WTK1 mutations were ranked using the SIFT. Low SIFT scores (<0.05) predict mutations with severe effects on protein function.
To determine if Wtk1 was sufficient to confer Pst resistance, the susceptible varieties T. aestivum cv. Fielder and T. durum cv. Kronos were transformed with a 9.1 kb genomic fragment that includes the complete Wtk1 coding and flanking regions. Expression of the Wtk1 transgene was detected in 17 out of 22 T0 transgenic plants and their progenies. In subsequent generations, the Wtk1 transgene co-segregated with the stripe rust resistance phenotype (
Three alternative transcript variants (Main isoform, Isoform2 and Isoform3,
Both WTK1 kinase-like domains belong to the Protein Kinase catalytic domain-like superfamily and display neither membrane-targeting motifs nor known receptor sequences (
A search for Wtk1 sequences in the chromosome arm 1BS of CS and Zavitan whole genome assemblies revealed the presence of non-functional alleles, designated as wtk1, in both of these susceptible lines. These alleles differ from the functional Wtk1 allele of G25 by indels (e.g. insertions of transposable elements such as Veju, Harbinger and MITE) that have changed the reading frame of exon 4 and generated premature stop codons in CS and Zavitan, relative to G25 (
Collinear orthologs of Wtk1 were found on all homologous group 1 chromosomes of tetraploid (Triticum turgidum ssp. dicoccoides, genome composition AABB) and hexaploid wheat (T. aestivum, AABBDD), as well as in diploid wheat relatives Triticum urartu (AA), Aegilops speltoides (SS), Aegilops tauschii (DD), representing the A, B, and D genomes, respectively, and in the 1R chromosome of rye (Secale cereale, RR). A phylogenetic analysis, conducted at the protein level, clustered all of these onto one branch, consisting of subclusters corresponding to the 1A, 1B and 1D copies (
380 Triticum accessions were evaluated for the presence of Wtk1 alleles. The functional Wtk1 allele was detected in 18% of the accessions of the Southern (Israel, Lebanon, Jordan, or Syria) DIC populations and not detected in the Northern (Turkey or Iran) populations (Tables 9 and 10) from which domesticated wheat is thought to have been derived. This suggests that Wtk1 was not incorporated into the initial domesticated forms. Diagnostic Kompetitive Allele Specific PCR (KASP) markers for each kinase-like domain of WTK1 (SEQ ID NOs: 107-109 for KinI and SEQ ID Nos: 110-112 for KinIII) were developed, and they can differentiate between functional (Wtk1) and non-functional (wtk1) alleles (Table 11,
1Accession sources are indicated in the Materials and Methods.
2Wild emmer wheat originated from Israel, Lebanon, Jordan, and Syria.
3Wild emmer wheat originated from Turkey, and Iran.
Triticum and Aegilops species evaluated for
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
dicoccum
Triticum
turgidum
carthlicum
Triticum
turgidum
carthlicum
Triticum
turgidum
polonicum
Triticum
turgidum
polonicum
Triticum
turgidum
polonicum
Triticum
turgidum
polonicum
Triticum
turgidum
polonicum
Triticum
monococcum
Triticum
monococcum
Triticum
monococcum
Triticum
monococcum
Triticum
monococcum
Triticum
speltoides
Triticum
speltoides
Triticum
speltoides
Triticum
speltoides
Triticum
speltoides
Triticum
speltoides
Triticum
speltoides
Triticum
speltoides
speltoides
Triticum
bicorne
Triticum
bicorne
Triticum
triunciale
Triticum
triunciale
Triticum
triunciale
Triticum
timopheevii
timopheevii
Triticum
timopheevii
timopheevii
Triticum
timopheevii
timopheevii
Triticum
longissimum
longissima
Triticum
longissimum
longissima
Triticum
longissimum
sharonensis
Triticum
longissimum
sharonensis
Triticum
longissimum
sharonensis
Triticum
longissimum
Triticum
longissimum
Triticum
squarrosum
Triticum
squarrosum
Triticum
squarrosum
Triticum
squarrosum
Triticum
cylindricum
Triticum
cylindricum
Triticum
crassum
Triticum
crassum
Triticum
ventricosum
Triticum
ventricosum
Triticum
ovatum
Triticum
ovatum
Triticum
triaristatum
Triticum
triaristatum
Triticum
triunciale
Triticum
kotschyi
Triticum
columnare
Triticum
ventricosum
Triticum
lorentii
Triticum
caudatum
Triticum
peregrinum
Aegilops
comosa
Aegilops
comosa
Aegilops
comosa
Aegilops
comosa
Aegilops
comosa
Aegilops
comosa
Aegilops
comosa
Aegilops
comosa
Aegilops
comosa
Aegilops
comosa
1Accession sources are indicated in the Materials and Methods.
Durum and common wheat accessions evaluated for the presence of
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
turgidum
durum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
Triticum
aestivum
1
T. durum lines from the Salamini collection.
2,3The pedigrees of the Yr15 introgression lines, as well as their recurrent lines (2T. turgidum sp. durum and 3T. aestivum) are known in the art.
Most of the previously cloned wheat resistance genes belong to the CC-NBS-LRR class. Therefore, the cloning of Yr15 encoding a protein with two tandem kinases broadens the tools available to diversify the sources of disease resistance in this economically important group of species. Moreover, the tandem kinases encoded by Yr15 are highly divergent from other tandem kinases involved in disease resistance in these species, including the barley stem rust resistance gene RPG1 and the true loose smut resistance candidate gene Un8.
The occurrence of functional resistance genes with a tandem kinase structure (WTK1 and RPG1), as well as of many WTK1 orthologs and paralogs in wheat and its near relatives, motivated us to search for similar protein architectures across the plant kingdom. Altogether, we found 92 predicted proteins that are composed of putative kinase and pseudokinase domains in tandem; like WTK1, none had additional conserved domains. Most of the putative kinase domains share key conserved residues (
The phylogenetic analysis showed that all 184 putative kinase and pseudokinase domains of the 92 predicted proteins could be sorted into 11 major clades and two singletons (
Some of the Yr15 introgression lines showed a partial resistance response when challenged with a few races that appeared in Europe more than a decade ago (e.g. Pst race DK92/02). However, we demonstrate here that pyramiding Yr15 with additional resistance genes, such as Yr5, provides full protection against races that are virulent or partially virulent on each one of them separately (
Microscopic observations of the interactions between host plant cells and invading pathogen structures showed substantially larger fungal colonies and much more abundant pathogen feeding structures in the susceptible common wheat cultivar Avocet S compared with its resistant (Avocet+Yr15) near isogenic line (NIL), 3-14 dpi (
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
This application claims the benefit of priority from U.S. Provisional Patent Application No. 62/575,670, filed Oct. 23, 2017, and U.S. Provisional Patent Application No. 62/575,688, filed Oct. 23, 2017, the contents of which are all incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2018/051081 | 10/3/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62575670 | Oct 2017 | US | |
| 62575688 | Oct 2017 | US |
