Cloning of wheat broad-spectrum powdery mildew-resistant gene pm26 and use thereof

Abstract

Cloning of a wheat broad-spectrum powdery mildew-resistant gene Pm26 and a use thereof. A Pm26 protein is a protein composed of the amino acid sequence represented by SEQ ID No. 3. Also provided is a gene Pm26, wherein a coding region is DNA molecules represented by positions 3001-9489 in SEQ ID No. 1, or the coding region is DNA molecules represented by SEQ ID No. 2. Further provided are gene mapping, map-based cloning and powdery mildew-resistant biological function identification methods for the wheat broad-spectrum powdery mildew-resistant gene PM26. The wheat broad-spectrum powdery mildew-resistant gene Pm26 can be widely used in plant fields of wheat disease-resistant genetic breeding, germplasm resource improvement, transgenic and genome editing breeding and the like, and plays an important role in improving and enhancing the germplasm resources of crops such as wheat.

Description

Cloning and application of wheat broad-spectrum powdery mildew resistance gene Pm26 Technical Field

[0001] The invention belongs to the field of crop molecular biology and molecular breeding, and particularly relates to the cloning of a wheat broad-spectrum powdery mildew resistance gene Pm26 and its application. Background Art

[0002] Wheat powdery mildew is a fungal disease caused by the wheat-specific form of the grass fungus Blumeria graminis f. sp. tritici. It is a major wheat disease worldwide, posing a significant challenge to wheat production safety. According to statistics from the National Agricultural Technology Extension Service Center, over the past decade, approximately 100 million mu (approximately 166 acres) of wheat powdery mildew-affected areas in my country have been affected, accounting for approximately one-fifth of all wheat diseases that year. Production practice has shown that, compared with spraying chemical pesticides, breeding and promoting disease-resistant varieties is the most economical, safe, and effective measure for controlling wheat powdery mildew. The key to developing disease-resistant varieties lies in identifying disease-resistant genes. To date, 69 powdery mildew resistance genes have been officially identified in common wheat and its related species. The discovery and utilization of these powdery mildew resistance genes has expanded the genetic resources for wheat disease resistance breeding and played a significant role in controlling wheat powdery mildew. However, the continuous mutation of new and virulent powdery mildew races has led to the reduction or loss of resistance in some genes. For example, Pm8 was once the most widely used powdery mildew resistance gene in my country. Its loss of resistance caused a major powdery mildew outbreak in the late 20th century. Currently, the powdery mildew resistance genes that remain effective in promoted wheat varieties in my country, or are effective in some regions, are primarily a few genes and their allelic variants, such as Pm2, Pm4, and Pm21. Therefore, cloning and utilizing powdery mildew resistance genes from diverse sources with good resistance is crucial for improving disease resistance breeding in Chinese wheat.

[0003] Wild emmer wheat (Triticum dicoccoides, AABB) is a wild relative of wheat and the direct ancestor of tetraploid and hexaploid wheat. Wild emmer wheat thrives in diverse habitats, undergoing complex environmental evolution over a long period of time, accumulating rich genetic diversity and harboring a rich reservoir of powdery mildew resistance genes. To date, eight officially named powdery mildew resistance genes have been discovered and mapped from wild emmer wheat: Pm16, Pm26, Pm30, Pm36, Pm41, Pm42, Pm64, and Pm69. With the exception of Pm41 and Pm69, no other genes have been cloned. The discovery and cloning of powdery mildew resistance genes in wild emmer wheat is of great significance for the genetic improvement of modern wheat varieties using modern genetic manipulation techniques, such as marker-assisted selection and gene editing, to exploit these resistance genes.

[0004] To date, several wheat powdery mildew resistance genes have been successfully cloned from wheat and its wild species, including Pm1, Pm2, Pm3, Pm4, Pm5, Pm8, Pm13, Pm17, Pm21, Pm24, Pm36, Pm38 / Lr34 / Yr18 / Sr57, Pm41, Pm46 / Lr67 / Yr46 / Sr55, Pm55, Pm57, Pm60, Pm69, and WTK4. More than half of these powdery mildew resistance genes encode typical NLR (Nucleotide-binding leucine-rich repeat)-type disease resistance proteins. NLR-type disease resistance proteins can specifically recognize pathogen effector factors and trigger a localized programmed cell death (programmed cell death) at the site of pathogen infection, known as the hypersensitive response (HR). This restricts the growth and reproduction of the pathogen, thereby protecting other parts of the plant from infection. SUMMARY OF THE INVENTION

[0005] The technical problem to be solved by the present invention is to provide a clone of a wheat broad-spectrum powdery mildew resistance gene Pm26 and an application thereof. Technical Solutions

[0006] In a first aspect, the present invention provides a protein, named Pm26 protein, which is the following A1) or A2) or A3):

[0007] A1) a protein comprising the amino acid residues shown in SEQ ID No. 3;

[0008] A2) a protein derived from A1) with the amino acid sequence of SEQ ID No. 3 having one or more amino acid residues substituted and / or deleted and / or added and having the same function, or a protein having more than 80% homology (identity) with the protein of A1);

[0009] A3) A fusion protein is obtained by adding a tag protein to the end of the protein shown in A1).

[0010] The above protein is derived from wheat.

[0011] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0012] In the above-mentioned proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, Myc tag, GST tag, and / or SUMO tag, etc.

[0013] The protein in A1) above, which is a protein consisting of the amino acid sequence shown in SEQ ID No. 3;

[0014] In the protein described in A2), the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0015] The gene encoding the protein in A2) above can be obtained by deleting one or more amino acid residue codons from the DNA sequence shown in SEQ ID No. 2, and / or performing missense mutations of one or more base pairs, and / or attaching a tag to the coding sequence at its 5' and / or 3' ends. In the above-mentioned protein, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, Advanced BLAST 2.1 can be used to calculate the identity of a pair of amino acid sequences by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0016] In the above proteins, the above 80% or greater identity may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0017] In a second aspect, the present invention provides a nucleic acid molecule encoding the protein of the first aspect.

[0018] The nucleic acid molecule described above, designated as gene Pm26, is located on wheat chromosome 2BS and is any one of the following DNA molecules B1) to B4):

[0019] B1) The coding region includes the DNA molecule shown in SEQ ID No. 1, positions 3001-9489;

[0020] B2) the coding region (CDS) includes the DNA molecule shown in SEQ ID No. 2;

[0021] B3) a DNA molecule that hybridizes under stringent conditions with the DNA sequence defined in B1) or B2) and encodes a protein having the same function;

[0022] B4) A DNA molecule that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous (identical) to the DNA sequence defined in B1) or B2) and encodes a protein having the same function.

[0023] In the nucleic acid molecule described above, B1) may be a DNA molecule whose coding region is shown in positions 3001-9489 of SEQ ID No. 1.

[0024] In the nucleic acid molecule described above, B2) may be a DNA molecule whose coding region is represented by SEQ ID No. 2.

[0025] Specifically, what is shown in B1) above is genomic DNA; what is shown in B2) above is cDNA.

[0026] The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" used in the present invention are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited to these. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and / or include a coding sequence in a cDNA, and / or include a cDNA and its regulatory sequences. In specific embodiments, such as with respect to an isolated nucleic acid sequence, it is preferably assumed to be cDNA. A person of ordinary skill in the art can easily use known methods, such as gene editing methods, to mutate the nucleotide sequence encoding the protein Pm26 of the present invention. Any artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the protein Pm26 isolated according to the present invention, as long as they encode the protein Pm26, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0027] The term "identity" refers to sequence similarity to a naturally occurring nucleic acid sequence. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences. The 90% or greater identity can mean at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0028] The above stringent conditions are hybridization and washing twice at 68°C in a 2×SSC, 0.1% SDS solution for 5 minutes each, and hybridization and washing twice at 68°C in a 0.5×SSC, 0.1% SDS solution for 15 minutes each; or hybridization and washing at 65°C in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution.

[0029] In a third aspect, the present invention provides a recombinant vector, an expression cassette or a recombinant bacterium containing the nucleic acid molecule described in the second aspect.

[0030] As described above, the expression cassette containing a nucleic acid molecule refers to a DNA sequence capable of expressing the aforementioned proteins in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary to express the nucleic acid molecule for any of the aforementioned proteins. The regulatory sequences are capable of directing the expression of any of the aforementioned proteins from the coding sequence in a suitable host cell under compatible conditions. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for ligating the regulatory sequences to the coding region of the protein-encoding nucleic acid sequence, the regulatory sequences may be provided with linkers. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence recognized by the host cell in which the nucleic acid sequence is to be expressed. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. A regulatory sequence may also be a suitable transcription terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that is functional in the selected host cell may be used in the present invention. A regulatory sequence may also be a suitable leader sequence, i.e., an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence may be operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the selected host cell may be used in the present invention. A regulatory sequence may also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of the protein that directs the encoded protein into the cell's secretory pathway. Any signal peptide coding region that directs the expressed protein into the secretory pathway of the selected host cell may be used in the present invention. It may also be desirable to add regulatory sequences that can regulate protein expression based on the growth conditions of the host cells. Examples of regulatory sequences are those that can turn gene expression on or off in response to chemical or physical stimuli (including in the presence of regulatory compounds). Other examples of regulatory sequences are those that enable gene amplification. In these instances, the protein-encoding nucleic acid sequence should be operably linked to the regulatory sequences.

[0031] Existing plant expression vectors can be used to construct a recombinant expression vector containing the protein encoding gene expression cassette.

[0032] When preparing an expression vector, a nucleic acid molecule encoding any of the above-mentioned proteins can be located in the vector so as to be operably linked to an appropriate expression control sequence. The recombinant expression vector can be any vector (e.g., a plasmid or virus) that is convenient for recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be a linear or closed-loop plasmid. The vector can be an autonomously replicating vector (i.e., a complete structure present outside the chromosome that can replicate independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is one that, when introduced into the host cell, will be integrated into the genome and replicated together with the chromosome into which it is integrated. In addition, a single vector or plasmid can be used, or two or more vectors or plasmids, or transposons, that collectively contain the entire DNA that will be introduced into the host cell genome can be used. The vector contains one or more selectable markers that facilitate the selection of transformed cells. A selectable marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or confers prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include the dal genes of Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. Vectors contain elements that enable stable integration of the vector into the host cell genome or autonomous replication of the vector within the cell, independent of the cellular genome. In the case of autonomous replication, the vector may also contain an origin of replication, enabling the vector to replicate autonomously in the target host cell. The origin of replication may contain a mutation that renders the vector temperature-sensitive in the host cell. More than one copy of a nucleic acid molecule encoding any of the above-described proteins of the present invention may be inserted into the host cell to increase the yield of the gene product. The copy number of the nucleic acid molecule can be increased by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or by inserting an amplifiable selectable marker along with the nucleic acid molecule. Cells containing amplified copies of the selectable marker gene, and thus the additional copies of the nucleic acid molecule, can be selected by culturing the cells in the presence of a suitable selective agent. The operations for ligating the above-mentioned elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0033] The term "operably linked" is defined herein as a configuration in which a regulatory sequence is appropriately positioned relative to the coding sequence of a DNA sequence such that the regulatory sequence directs the expression of a protein.

[0034] In the above-mentioned biological materials, the recombinant bacteria can specifically be yeast, bacteria, algae or fungi.

[0035] In a fourth aspect, the present invention provides use of the protein described in the first aspect, the nucleic acid molecule described in the second aspect, or the recombinant vector, expression cassette or recombinant bacteria described in the third aspect in regulating plant resistance to powdery mildew.

[0036] In the above, the regulating plant resistance to powdery mildew is to improve the plant resistance to powdery mildew.

[0037] The regulation may be at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) regulation of the post-translational activity of the gene (that is, regulation of the activity of the protein translated from the gene).

[0038] In a fifth aspect, the present invention provides use of the protein described in the first aspect, the nucleic acid molecule described in the second aspect, or the recombinant vector, expression cassette or recombinant bacteria described in the third aspect in cultivating powdery mildew resistant plants.

[0039] In a sixth aspect, the present invention provides the use of a substance that inhibits the biological function (activity) of the protein described in the first aspect or a substance that inhibits the expression of the nucleic acid molecule described in the second aspect in reducing plant resistance to powdery mildew.

[0040] In the seventh aspect, the use of a substance that inhibits the biological function of the protein described in the first aspect or a substance that inhibits the expression of the nucleic acid molecule described in the second aspect in cultivating powdery mildew-sensitive plants.

[0041] In the applications described above, the inhibition of the expression of the nucleic acid molecule described in the second aspect can be achieved by gene knockout or gene silencing.

[0042] Gene knockout refers to the inactivation of a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene through changes in the DNA sequence.

[0043] Gene silencing refers to the phenomenon of suppressing or under-expressing a gene without damaging the original DNA. Gene silencing, predicated on not altering the DNA sequence, results in suppressing or under-expressing the gene. Gene silencing can occur at two levels: transcriptional gene silencing due to factors such as DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene after transcription through specific inhibition of target RNA. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational inhibition.

[0044] In the applications described above, the substance that inhibits the biological function (activity) of the protein described in the first aspect or the substance that inhibits the expression of the nucleic acid molecule described in the second aspect may be an agent that inhibits or reduces the expression of the nucleic acid molecule. The agent that inhibits or reduces the expression of the nucleic acid molecule may include, for example, an agent that knocks out the gene via homologous recombination or an agent that knocks out the gene via CRISPR / Cas9. The agent that inhibits or reduces the expression of the nucleic acid molecule may comprise a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0045] Furthermore, the substance that inhibits the biological function (activity) of the protein described in the first aspect or the substance that inhibits the expression of the nucleic acid molecule described in the second aspect is specifically a BSMV-VIGS system, including pCaBS-α, pCaBS-β, and pCaBS-γbLIC-Pm26. Among them, pCaBS-γbLIC-Pm26 is a silencing vector constructed by ligating the target fragment shown in SEQ ID No. 4 with the pCaBS-γbLIC vector.

[0046] In an eighth aspect, the present invention provides a method for cultivating a transgenic plant resistant to powdery mildew, the method comprising the steps of: increasing the content, biological function and / or activity of the protein of the first aspect in a starting plant to obtain a transgenic plant;

[0047] The transgenic plants have higher resistance to powdery mildew than the starting plants.

[0048] In the method described above, the increasing of the content, biological function and / or activity of the protein described in the first aspect in the starting plant, or the increasing of the expression of the nucleic acid encoding the protein described in the first aspect in the starting plant, is to introduce the nucleic acid encoding the protein into the starting plant.

[0049] The "increasing the content, biological function and / or activity of the protein described in the first aspect in the starting plant" can be achieved by expressing or overexpressing the protein, or increasing the activity of the protein, through methods well known in the art such as multiple copies, changing promoters, regulatory factors, and transgenics.

[0050] In a ninth aspect, the present invention provides a method for cultivating a transgenic plant resistant to powdery mildew, the method comprising the steps of: increasing the expression of a nucleic acid molecule encoding the protein of the first aspect in a starting plant to obtain a transgenic plant;

[0051] The transgenic plants have higher resistance to powdery mildew than the starting plants.

[0052] In the above-mentioned method, the "increasing the expression of the nucleic acid molecule encoding the protein of the first aspect in the starting plant" can specifically be introducing the nucleic acid molecule encoding the protein into the starting plant.

[0053] In the above, the “introducing the nucleic acid expressing the protein into the starting plant” refers to introducing the nucleic acid expressing the protein into the starting plant via a recombinant expression vector; the recombinant expression vector may specifically be the pTPCK303-OE3-Pm26 vector mentioned in the examples.

[0054] In a tenth aspect, the present invention provides a method for cultivating a transgenic plant susceptible to powdery mildew, the method comprising the steps of: reducing the content, biological function and / or activity of the protein of the first aspect in a starting plant to obtain a transgenic plant;

[0055] The transgenic plants have lower resistance to powdery mildew than the starting plants.

[0056] In an eleventh aspect, the present invention provides a method for cultivating a transgenic plant susceptible to powdery mildew, the method comprising the steps of: inhibiting the expression of a nucleic acid molecule encoding the protein of the first aspect in a starting plant to obtain a transgenic plant;

[0057] The transgenic plants have lower resistance to powdery mildew than the starting plants.

[0058] In the eighth to eleventh aspects above, the starting plant contains a gene encoding the protein described in the first aspect.

[0059] In the above, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0060] The monocotyledonous plant is a grass plant.

[0061] The grass plant is a plant of the genus Triticeae;

[0062] The Triticum plant includes but is not limited to wheat.

[0063] In a twelfth aspect, the present invention provides a primer pair for amplifying the full length or a fragment of the nucleic acid molecule described in the second aspect.

[0064] In an embodiment of the present invention, the primer pair is 666COM-1F / 1R and 666COM-2F / 2R, or 666OE-1F / 1R.

[0065] The present invention provides a BSMV-VIGS vector containing the Pm26 gene, which is constructed using the method reported in the literature (Yuan C, Li C, Yan L, et al. A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One. 2011;6(10):e26468). The nucleotide sequence of the Pm26 gene fragment used to construct the BSMV-VIGS vector is shown in SEQ ID No. 4.

[0066] The present invention provides a complementary expression vector and an overexpression vector of the Pm26 gene, which are constructed by the method reported in the reference literature (Lu et al. A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nat. Commun. 2020; 11, 680). Beneficial effects

[0067] The present invention provides methods for mapping and cloning the wheat broad-spectrum powdery mildew resistance gene Pm26, as well as identifying its biological function in powdery mildew resistance. The wheat broad-spectrum powdery mildew resistance gene Pm26 can be widely used in plant breeding, germplasm improvement, transgenic breeding, and genome editing, among other areas. It plays an important role in improving and enhancing the germplasm resources of crops like wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 shows the identification of multiple races of powdery mildew fungus in wheat lines Pm26-40 and IW170.

[0069] Figure 2 shows the map-based cloning of the wheat powdery mildew resistance gene Pm26.

[0070] Figure 3 is the EMS mutant verifying the powdery mildew resistance function of Pm26.

[0071] Figure 4 shows the BSMV-VIGS verification of the powdery mildew resistance of Pm26.

[0072] Figure 5 shows the transgenic verification of the powdery mildew resistance of Pm26. Modes for Carrying Out the Invention

[0073] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0074] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0075] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0076] The powdery mildew strain E09 described in the following examples is preserved in this laboratory and is described in the literature "Li, M. et al. A CNL protein in wild emmer wheat confers powdery mildew resistance. New Phytol. 228, 1027-1037 (2020). The public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials are only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0077] In the following examples, the method for identifying powdery mildew resistance in wheat seedlings was specifically referred to the following document: Liu ZY, Sun QX, Ni ZF, Yang TM (1999) Development of SCAR markers linked to the Pm21 gene conferring resistance to powdery mildew in common wheat. Plant Breeding 118: 215-219.

[0078] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0079]

[0080]

[0081] Example 1. Discovery and cloning of the wheat broad-spectrum powdery mildew resistance gene Pm26

[0082] 1. Powdery mildew resistance identification

[0083] The common hexaploid wheat material Pm26-40 (described in the following reference: Zhang Deyun. Construction of a physical map of the powdery mildew resistance genes MlIW170, Pm26, MlWE74, and PmWE35 in wild emmer wheat. (PhD thesis), 2018) is an introgression line containing a fragment of the powdery mildew resistance gene Pm26, obtained by crossing the wild emmer wheat material TTD140 with the common wheat line Chinese Spring (CS) through multiple generations of backcrossing and selfing. It was kindly provided by Professor Rong Junkang of Zhejiang Agricultural and Forestry University. The highly powdery mildew-resistant wild emmer wheat material IW170 was kindly provided by Professor Eviatar Nevo of the Institute of Evolution, University of Haifa, Israel. The common wheat lines Chinese Spring and durum wheat 81086A served as the corresponding powdery mildew-susceptible parents. Thirty-six powdery mildew races collected from various locations across China were used to identify the multiple races of wheat lines Pm26-40 and IW170 by in vitro inoculation at the seedling stage. Pm26-40 showed high resistance to 28 powdery mildew species; IW170 showed high resistance to all 36 powdery mildew species, making it an excellent broad-spectrum powdery mildew resistant material (Figure 1).

[0084] 2. Fine mapping of the wheat broad-spectrum powdery mildew resistance gene Pm26

[0085] Disease resistance at the seedling stage was assessed using powdery mildew race E09, a fungus prevalent in Beijing, in F1 and F2 segregating populations and F3 families of wheat lines Pm26-40, CS, IW170, 81086A, Pm26-40 / CS, and IW170 / 81086A. The results showed that Pm26-40 and IW170 exhibited immune responses (IT = 0), while CS and 81086A exhibited highly susceptible responses (IT = 4). F1 lines of CS × Pm26-40 and 81086A × IW170 exhibited highly susceptible responses (IT = 3) (Figure 2a). F2 and F3 lines of Pm26-40 / CS were tested. 2:3 The results of the seedling powdery mildew identification showed that among the 77 Pm26-40 / CS F2 segregating populations, 21 showed resistance and 56 showed susceptible. The chi-square test was consistent with the segregation ratio of 1:3 recessive single gene inheritance. 2:3Among the families, 45 showed homozygous resistance, 110 showed segregation, and 50 showed homozygous susceptibility. The chi-square test was consistent with the segregation ratio of 1:2:1 recessive single gene inheritance. The above studies indicate that the powdery mildew resistance gene in Pm26-40 is controlled by the recessive single gene Pm26. 2:3 The results of the powdery mildew identification of the family at seedling stage showed that 30 of the 130 F2 segregating populations were resistant to the disease and 100 were susceptible to the disease. The chi-square test was consistent with the segregation ratio of 1:3 recessive single gene inheritance. 2:3 Among the families, 43 showed homozygous resistance, 105 showed segregation, and 45 showed homozygous susceptibility. The chi-square test showed a segregation ratio of 1:2:1, consistent with recessive single-gene inheritance. This indicates that the powdery mildew resistance gene in IW170 is controlled by a recessive single gene, MlIW170 (Table 2).

[0086]

[0087] In 2000, Dr. Junkang Rong discovered and located a powdery mildew resistance gene from wild emmer wheat and named it Pm26. It is located at the end of chromosome 2BS and co-segregates with the molecular marker Xwg516 (Rong JK, Millet E, Manisterski J, et al. A new powdery mildew resistance gene: introgression from wild emmer into common wheat and RFLP-based mapping. Euphytica. 2000; 115, 121-126). In 2012, Dr. Liu Ziji discovered a powdery mildew resistance gene from wild emmer wheat, tentatively named MlIW170. It was also located at the end of chromosome 2BS, 2.69 cM away from markers CJ945509 and BQ169830, and co-segregated with marker Xcau516, ​​which was developed based on marker Xwg516 (Liu ZJ, Zhu J, Cui Y, et al. Identification and comparative mapping of a powdery mildew resistance gene derived from wild emmer (Triticum turgidum var. dicoccoides) on chromosome 2BS. Theor Appl Genet. 2012; 124, 1041-1049). In 2015, Dr. Liang Yong screened the BAC library of wild emmer wheat TZ-2 and constructed the physical map of MlIW170, and found that MlIW170 was located in the R gene cluster (Liang Y, Zhang DY, Ouyang SH, et al. Dynamic evolution of resistance gene analogs in the orthologous genomic regions of powdery mildew resistance gene MlIW170inTriticum dicoccoidesandAegilops tauschii. Theor Appl Genet.2015, 128(8),1617-1629).Previous studies have shown that the Pm26 locus is located at the terminal region of chromosome 2BS (Figure 2b). Molecular markers were developed based on the reference genome sequence of wild emmer wheat (Zavitan) corresponding to the locus. Polymorphic markers WGGBH553, Xcau516, ​​WGGBD412, WGGBH346, WGGD698, WGGD649, and WGGBD560 were found to be tightly linked to the Pm26 gene. Using the common SSR markers Xcau357 and Xwmc243 flanking the locus, 4664 F2 segregating populations of Pm26-40 / CS and 4388 F2 segregating populations of IW170 / 81086A were screened. A total of 53 independent crossovers were identified in the Pm26-40 / CS F2 population and 62 in the IW170 / 81086 population. The molecular markers within the localization interval were used to identify the genotype of the exchanged plants, and the Pm26 gene was precisely located in combination with the phenotypic data. Pm26 was ultimately located between the molecular markers WGGBD412 and WGGBH346, with a genetic interval of 0.18 cM corresponding to the Pm26-40 / CS population (Figure 2c), a genetic interval of 0.07 cM corresponding to the IW170 / 81086A population (Figure 2d), and an 800 kb physical interval corresponding to the wild emmer wheat Zavitan V2.0 reference genome. A total of eight genes were annotated, including two phosphoglycerate mutase genes (TRIDC2BG005010 and TRIDC2BG005040) and six disease resistance-related CC-NBS-LRR genes (TRIDC2BG005090-1, TRIDC2BG005100, TRIDC2BG005090-2, TRIDC2BG005110, TRIDC0UG006500, and TRIDC2BG005230) (Figure 2e). The above results showed that the powdery mildew resistance gene Pm26 was located in the disease resistance R gene cluster.

[0088] To obtain the physical map of the Pm26 locus, the genome of wild emmer wheat IW170 was sequenced and assembled using the PacBio HiFi third-generation genome sequencing strategy. The sequencing results are summarized as follows (Table 3).

[0089]

[0090] Four cells were used, generating an average of 35.35 Gb of data per cell, for a total of 141.4 Gb of data, covering the wild emmer genome at a sequencing depth of approximately 14x. After sequence assembly, 3208 contigs were obtained with an N50 of 9.4 Mb. These 3208 contigs were aligned using the molecular markers WGGBD412 and WGGBH346, which flank the MlIW170 gene. The resulting contig, ptg000834l, spans the localized region, with a total length of 4.1 Mb. The physical distance within the localized region where Pm26 is located is 909 kb (Figure 2f).

[0091] 3. Map-based cloning of the wheat broad-spectrum powdery mildew resistance gene Pm26

[0092] A mutant library was created by treating seeds of the disease-resistant parents, Pm26-40 and IW170, with EMS mutagenesis. 1,000 M2 families were obtained from Pm26-40 and 3,300 M2 families were obtained from IW170. These mutant families were identified for resistance to powdery mildew at the seedling stage in the greenhouse using the powdery mildew race E09. Susceptible mutants were then transplanted to obtain homozygous susceptible mutants. A total of five homozygous susceptible mutants were obtained in the Pm26-40 background and nine in the IW170 background (Figure 3a). These nine IW170 susceptible mutants and the wild-type IW170 were then subjected to seedling RNA-Seq.

[0093] Transcriptome data from the wild-type IW170 and nine mutants were anchored to the IW170 sequencing-assembled genome. Results showed that six of the nine candidate genes within the mapped interval were expressed, namely, CNL1-CNL6. The CNL1 gene contained point mutations in the RNA-Seq results of the IW170 mutants, resulting in amino acid changes. The CNL1 genes of mutants M452, M3200, M1401, M2279, and M1241 exhibited SNPs that resulted in amino acid changes compared to the wild-type IW170 (Figure 3b). Therefore, CNL1 was selected as a candidate gene for Pm26 for further analysis.

[0094] Based on the sequence information from IW170 sequencing and genome assembly, specific primers were designed to amplify the Pm26 gene in four parental materials: Pm26-40, CS, IW170, and 81086A. The Pm26 gene was amplified only in the two disease-resistant parents, Pm26-40 and IW170; no amplified bands were found in CS and 81086A. Sanger sequencing revealed that the Pm26 gene (denoted as CNL1 in the figure) was completely identical in sequence in Pm26-40 and IW170 (Figure 2g). Annotation of the NLR protein encoded by the Pm26 gene using NCBI revealed that Pm26 contains the CC, NB-ARC, and LRR domains typical of disease-resistant R proteins. Furthermore, an atypical NCKX domain is integrated between the CC and NB-ARC domains (Figure 3b). A dominant marker WGGBM24 was designed based on the presence or absence of Pm26 in the parents. After verification by exchanging individual plants in the Pm26-40 / CS segregation population and the IW170 / 81086A segregation population, WGGBM24 co-segregated with the phenotype (Figure 2c, d).

[0095] Sanger sequencing of the Pm26 sequence of all mutants in the Pm26-40 and IW170 backgrounds revealed that among the nine homozygous susceptible mutants in the IW170 background, nine harbored mutations in the Pm26 gene. One of these mutations was a 6-bp deletion resulting in a two-amino acid deletion, and eight were G / CA / T variants resulting in nonsynonymous amino acid changes. Among the five homozygous susceptible mutants in the Pm26-40 background, five harbored mutations in Pm26, including one G / CA / T variant leading to a premature termination and four G / CA / T variants resulting in nonsynonymous amino acid changes (Figure 3b, c). These results suggest that Pm26 confers powdery mildew resistance in Pm26-40 and IW170. Sequence analysis and allelic testing confirmed that Pm26 and MlIW170 share the same Pm26 gene.

[0096] After sequencing, the Pm26 gene is located on the wheat chromosome 2BS. Its genomic sequence is shown in SEQ ID No.1, 3001-9489, its cDNA sequence is shown in SEQ ID No.2, and its amino acid sequence is shown in SEQ ID No.3.

[0097] Positions 1-3000 of SEQ ID No. 1 are the upstream promoter region, positions 3001-9489 are the gene region, and positions 9490-12690 are the gene downstream regulatory region.

[0098] Example 2: Functional verification of the Pm26 gene

[0099] 1. BSMV-VIGS technology silencing Pm26 gene

[0100] To verify whether Pm26 has powdery mildew resistance, the Pm26 gene in Pm26-40 and IW170 was silenced using the previously reported BSMV-VIGS system for silencing endogenous genes in wheat (pCaBS-α, pCaBS-β, and pCaBS-γbLIC used are all described in the following literature: Yuan C, Li C, Yan L, et al. A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One. 2011;6(10):e26468).

[0101] The gene silencing vectors pCaBS-α, pCaBS-β, and pCaBS-γbLIC were kindly provided by Professor Li Dawei of China Agricultural University. The powdery mildew strain E09 was maintained in our laboratory.

[0102] 1. Construction of silencing vector

[0103] Using Pm26-VIGSF / Pm26-VIGSR (Table 1) as primers and the Pm26 gene shown in SEQ ID No. 1 as a template, the target fragment (SEQ ID No. 4) was obtained.

[0104] The pCaBS-γbLIC vector was then digested with the restriction endonuclease Apa I and the linearized product was recovered. The target fragment shown in SEQ ID No. 4 was then ligated with the pCaBS-γbLIC vector using the LIC site ligation principle to construct the silencing vector pCaBS-γbLIC-Pm26.

[0105] After sequencing, the silencing vector pCaBS-γbLIC-Pm26 was obtained by inserting the target fragment shown in SEQ ID No. 4 into the ApaⅠ restriction site of the pCaBS-γbLIC vector.

[0106] The silencing vector pCaBS-γbLIC-Pm26 was transformed into Agrobacterium EHA105 to obtain the recombinant bacterium EHA105 / pCaBS-γbLIC-Pm26.

[0107] 2. BSMV-VIGS induces Pm26 gene silencing, causing plants to be susceptible to powdery mildew

[0108] 1) After sowing, place N. benthamiana plants in a 20-22°C incubator with 16 hours of light and 8 hours of darkness. Grow them to the 6-8 leaf stage for BSMV-VIGS experiments.

[0109] 2) After activating the recombinant EHA105 / pCaBS-γbLIC-Pm26 obtained in step 1 above for 36-48 hours, a single clone was picked and inoculated into 1 mL of LB liquid medium (Kan+Rif) and cultured in a shaking incubator at 28°C and 220 rpm for 24 hours.

[0110] 3) Inoculate the culture medium into 10 mL LB liquid medium (Kan+Rif) containing 20 μM AS and 100 μM MES at a ratio of 1:100, and culture at 28°C, 200 rpm, and shake for 12 h to obtain a bacterial solution.

[0111] 4) Centrifuge the bacterial solution at 6000 rpm for 5 min to collect the cells, and resuspend the cells in an equal volume of tobacco infection solution (10mM MgCl2, 10mM MES, pH=5.2, 0.1Mm AS).

[0112] 5) Adjust the bacterial solution concentration to OD 600 =0.7, the bacterial solutions containing the pCaBS-α, pCaBS-β, and pCaBS-γbLIC vectors (recombinant bacteria obtained by transferring each vector into Agrobacterium EHA105) were mixed at a ratio of 1:1:1, or the bacterial solutions containing the pCaBS-α, pCaBS-β, and pCaBS-γbLIC-Pm26 vectors (recombinant bacteria obtained by transferring each vector into Agrobacterium EHA105) were mixed at a ratio of 1:1:1, and after standing at 28°C for 3-5h, they were injected into expanded leaves of Nicotiana benthamiana at the 6-8 leaf stage and labeled as BSMV:EV and BSMV:Pm26, respectively.

[0113] 6) 7-12 days after injection, the injected leaves and the first leaf above them were collected, fully ground in PBS (PH=7.2) buffer, and the juice was friction-inoculated into the first leaf of the one-leaf-one-heart Pm26-40 and IW170 wheat.

[0114] 7) Three days after inoculation, observe the virus expansion symptoms on the wheat leaves and inoculate powdery mildew E09 for disease resistance identification.

[0115] The results showed that 10 days after inoculation with powdery mildew E09, the leaves of Pm26-40 and IW170 inoculated with BSMV:Pm26 were susceptible to powdery mildew (Figure 4b, c).

[0116] B. qRT-PCR detection

[0117] Ten days after inoculation with the powdery mildew fungus E09 in step 7), genomic DNA from leaves of Pm26-40 and IW170 wheat susceptible to powdery mildew with white spots was extracted and PCR amplified using primers Pm26-M4.

[0118] The results are shown in Figure 4a. It can be seen that in the powdery mildew-susceptible Pm26-40 and IW170 plants, inoculation with BSMV: Pm26 can cause a significant downregulation of the expression of the Pm26 gene compared with the control BSMV: EV.

[0119] The above results showed that silencing the expression of Pm26 gene in Pm26-40 and IW170 could make the plants susceptible to powdery mildew.

[0120] 2. Transgenic verification of Pm26's powdery mildew resistance

[0121] To further confirm the powdery mildew resistance of Pm26, the recombinant expression vectors pCAMBIA1300-Pm26 and pTPCK303-OE3-Pm26 were introduced into the recipient wheat cultivar Fielder using Agrobacterium-mediated genetic transformation (Ishida Y, Tsunashima M, Hiei Y, Komari T. 2015. Wheat (Triticum aestivum L.) transformation using immature embryos. In: Wang K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol. 1223, pp189-198. Springer, New York, NY.) to verify whether Pm26 fully confers powdery mildew resistance. The pCAMBIA1300 plasmid, used as the base vector for constructing the complementation transgene, and the pTPCK303-OE3 plasmid, used as the base vector for constructing the overexpression transgene, were kindly provided by Professor Li Genying of the Shandong Academy of Agricultural Sciences.

[0122] 1. Construction of recombinant vector

[0123] 1) Construction of recombinant plasmid pCAMBIA1300-Pm26

[0124] (1) Take the pCAMBIA1300 plasmid (described in the following literature: Lu et al. A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nat. Commun. 2020; 11, 680), double-digest it with the restriction endonucleases BamHI and HindIII, and recover the vector backbone.

[0125] (2) Using the DNA fragment shown in SEQ ID No. 1 as a template, PCR amplification was performed using 666COM-1F / 1R and 666COM-2F / 2R (see Table 1) to obtain two PCR amplification products.

[0126] (3) The two PCR amplification products obtained in step (2) and the vector backbone recovered in step (1) were homologously recombined using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) to obtain the recombinant plasmid pCAMBIA1300-Pm26.

[0127] The recombinant plasmid pCAMBIA1300-Pm26 is obtained by homologously recombining the DNA fragment shown in SEQ ID No. 1 into the pCAMBIA1300 vector, and the expression of the Pm26 gene is driven by its own promoter (SEQ ID No. 1, positions 1-3000 from the 5' end).

[0128] 2) Construction of recombinant plasmid pTPCK303-OE3-Pm26

[0129] (1) Take the pTPCK303-OE3 plasmid (described in the following literature: Lu et al. A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nat. Commun. 2020; 11, 680), double-digest it with the restriction endonucleases BamHI and Sac1, and recover the vector backbone.

[0130] (2) Using the DNA fragment shown in SEQ ID No. 2 as a template, PCR amplification was performed using 666OE-1F / 1R (see Table 1) to obtain the amplified product.

[0131] (3) The PCR amplification product obtained in step (2) and the vector backbone recovered in step (1) were homologously recombined using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) to obtain the recombinant plasmid pTPCK303-OE3-Pm26.

[0132] The recombinant plasmid pTPCK303-OE3-Pm26 is a plasmid obtained by homologously recombining the DNA fragment shown in SEQ ID No. 2 into the pTPCK303-OE3 vector, and the Pm26 gene is driven by the Ubiquitin promoter of maize.

[0133] 2. Obtaining recombinant Agrobacterium

[0134] 1) The recombinant plasmid pCAMBIA1300-Pm26 was introduced into Agrobacterium tumefaciens EHA105 by heat shock transformation to obtain recombinant Agrobacterium, which was named EHA105 / pCAMBIA1300-Pm26.

[0135] 2) The recombinant plasmid pTPCK303-OE3-Pm26 was introduced into Agrobacterium tumefaciens EHA105 by heat shock transformation to obtain recombinant Agrobacterium, which was named EHA105 / pTPCK303-OE3-Pm26.

[0136] 3. Obtaining transgenic wheat with Pm26 gene

[0137] 1) Agrobacterium-mediated genetic transformation was used to transform EHA105 / pCAMBIA1300-Pm26 into the common wheat material Fielder, which is highly susceptible to powdery mildew. Complementary transgenic T0 plants were then obtained. After molecular identification, two positive plants were obtained, named COM-1 to COM-2.

[0138] The above molecular identification: genomic DNA was extracted from plant leaves, and PCR amplification was performed using WGGBM24 primers, and the 870 bp obtained was positive.

[0139] 2) Agrobacterium-mediated genetic transformation was used to transform EHA105 / pTPCK303-Pm26 into the common wheat material Fielder, which is highly susceptible to powdery mildew. Overexpression transgenic T0 plants were then obtained. After molecular identification, a total of three positive plants were obtained and named OE-1 to OE-3.

[0140] The above molecular identification: RNA was extracted from plant leaves, reverse transcribed to obtain cDNA, and PCR amplified using WGGBM24 primers, and the 870 bp obtained was positive.

[0141] 4. Powdery mildew resistance identification

[0142] The wheat seedling powdery mildew resistance identification method was used to test wheat powdery mildew resistance. The following phenotypic identification materials are Fielder, OE-1, OE-2, OE-3, COM-1, and COM-2. The specific method is as follows:

[0143] The wheat materials were sown and cultivated, and then tested for powdery mildew resistance at the seedling stage. The specific method was as follows: One week before planting the phenotypic test materials, saplings were planted and inoculated with the powdery mildew fungus E09 to fully infect the wheat seedlings, ensuring sufficient powdery mildew infestation under laboratory conditions. Each phenotypic test material was planted in a plug tray, with 15 seeds per hole. When the seedlings reached the one-leaf, one-heart stage, susceptible control saplings were placed around the trays and inoculated using both natural spread and manual dusting. Resistance was recorded 15 days after inoculation. The response was categorized into six levels based on the distribution of powdery mildew colonies on the leaves and the size of the lesions: 0 (immune), 0 (hypersensitive necrosis), 1 (highly resistant), 2 (moderately resistant), 3 (moderately susceptible), and 4 (highly susceptible), with levels 0-2 indicating resistance and 3-4 indicating susceptible.

[0144] The results are shown in FIG5 , which shows that, compared with Fielder, the two Pm26 complemented transgenic T0 families and the three CNL gene overexpression T0 families all showed immunity to powdery mildew.

[0145] The above results indicate that the Pm26 gene has good powdery mildew resistance function in common hexaploid wheat.

[0146] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0147] CROSS-REFERENCE TO RELATED APPLICATIONS

[0148] This application claims priority to the Chinese patent application (application number 202311536905.6) filed on November 17, 2023, the entire contents of which are incorporated herein by reference. Industrial Applicability

[0149] The wheat broad-spectrum powdery mildew resistance gene Pm26 of the present invention can be widely used in plant fields such as wheat disease resistance genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, and plays an important role in improving and modifying the germplasm resources of crops such as wheat.

Claims

1. A protein, which is (1) or (2): 1) A protein consisting of the amino acid sequence shown in SEQ ID No. 3; 2) A protein derived from 1) with the same function as that of SEQ ID No. 3, wherein one or more amino acid residues are substituted and / or deleted and / or added.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule of any one of the following 1)-4): 1) The coding region is the DNA molecule shown in positions 3001-9489 of SEQ ID No.1; 2) The coding region is the DNA molecule shown in SEQ ID No. 2; 3) A DNA molecule that hybridizes with the DNA sequence defined in 1) or 2) under stringent conditions and encodes a protein with the same function; 4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in 1) or 2) and encodes a protein with the same function.

4. A recombinant vector, expression cassette or recombinant bacterium containing the nucleic acid molecule according to claim 2 or 3.

5. Use of the protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the recombinant vector, expression cassette or recombinant bacterium according to claim 4 in regulating plant resistance to powdery mildew.

6. The use according to claim 5, characterized in that: The regulating plant resistance to powdery mildew is to improve the plant resistance to powdery mildew.

7. Use of the protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the recombinant vector, expression cassette or recombinant bacterium according to claim 4 in cultivating powdery mildew resistant plants.

8. Use of a substance that inhibits the biological function of the protein according to claim 1 or a substance that inhibits the expression of the nucleic acid molecule according to claim 2 or 3 in reducing plant resistance to powdery mildew.

9. Use of a substance that inhibits the biological function of the protein according to claim 1 or a substance that inhibits the expression of the nucleic acid molecule according to claim 2 or 3 in cultivating powdery mildew-sensitive plants.

10. A method for cultivating transgenic plants resistant to powdery mildew, comprising the following steps: increasing the content, biological function and / or activity of the protein of claim 1 in a starting plant to obtain a transgenic plant; The transgenic plants have higher resistance to powdery mildew than the starting plants.

11. A method for cultivating transgenic plants resistant to powdery mildew, comprising the following steps: increasing the expression of a nucleic acid molecule encoding the protein of claim 1 in a starting plant to obtain a transgenic plant; The transgenic plants have higher resistance to powdery mildew than the starting plants.

12. A method for cultivating transgenic plants susceptible to powdery mildew, comprising the following steps: reducing the content, biological function and / or activity of the protein of claim 1 in a starting plant to obtain a transgenic plant; The transgenic plants have lower powdery mildew resistance than the starting plants.

13. A method for cultivating transgenic plants susceptible to powdery mildew, comprising the following steps: inhibiting the expression of a nucleic acid molecule encoding the protein of claim 1 in a starting plant to obtain a transgenic plant; The transgenic plants have lower powdery mildew resistance than the starting plants.

14. A primer pair for amplifying the full length or a fragment of the nucleic acid molecule according to claim 2 or 3.

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