Late blight resistance gene, biomaterial, and use

Abstract

The present invention relates to the technical fields of gene segregation and plant improvement, and in particular to a late blight resistance gene, a biomaterial, and a use. An Rpi-caj1 nucleic acid molecule comprises: a nucleotide sequence as shown in any one of SEQ ID NOs: 1-6; a nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 7-12; a nucleotide sequence shown in any one of SEQ ID NOs: 13-18; and a nucleotide sequence having at least 75% sequence identity with the described nucleotide sequences. An important gene resource is provided for late blight resistance breeding of potatoes, thereby solving the problem of loss of disease resistance of potato varieties and providing a new guarantee for persistent disease resistance of potatoes.

Description

Late blight resistance genes, biomaterials and their applications Technical Field

[0001] The present application relates to the technical field of gene isolation and plant improvement, and in particular to late blight resistance genes, biomaterials and applications. Background Art

[0002] Potato late blight is internationally recognized as the most serious disease affecting potato production. Its outbreaks, once known as the Great Irish Famine, have posed a significant challenge to scientists worldwide for over a century. Due to the high virulence and mutagenicity of the late blight pathogen, it can quickly overcome the resistance of most existing potato varieties. This increasingly problematic loss of resistance poses a serious threat to potato production. As the world's largest potato producer, my country relies heavily on chemical pesticides for late blight control. The extensive use of these pesticides not only increases production costs and pollutes the environment, but also poses a threat to public health. Furthermore, the long-term and extensive use of the fungicide metalaxyl has led to the continuous mutation of late blight races, resulting in the development of resistance. Therefore, breeding and planting varieties with durable late blight resistance are effective alternatives for combating the disease. The identification of new broad-spectrum resistance genes and the appropriate aggregation of multiple resistance genes are currently key priorities in potato breeding for disease resistance.

[0003] The discovery and cloning of new broad-spectrum resistance genes for potato is a hot topic in late blight research. In early research on late blight resistance, the primary resistance genes came primarily from the wild hexaploid Mexican species, Solanum demissum. The 11 late blight resistance genes (R1-R11) contained in this species were all race-specific, major resistance genes. However, with the continuous variation of the physiological races of Phytophthora infestans, these early resistance genes have all lost their resistance properties. With the rapid development of modern molecular biology and high-throughput sequencing technology, potato breeders in Europe and the United States have continuously cloned new disease-resistance genes from various wild potato resources using methods such as disease-resistance gene map-based cloning combined with comparative genomics, resistance gene enrichment sequencing (Renseq), and pathogen effector omics. The most famous broad-spectrum resistance genes include Rpi-vnt1.1, Rpi-blb1, R8, Rpi-amr1, etc., but physiological subspecies of pathogenic Phytophthora that can overcome these disease-resistance genes have also emerged. Therefore, it is urgent to continue to discover and clone new broad-spectrum resistance genes for potatoes.

[0004] Summary of the Invention

[0005] In view of this, the present application provides a new late blight resistance gene, biomaterial and application. The present application provides a newly discovered late blight resistance gene Rpi-caj1, which provides an important gene resource for potato late blight resistance breeding.

[0006] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0007] The present application provides an Rpi-caj1 nucleic acid molecule comprising at least one of the following nucleotide sequences:

[0008] (a1) the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 6;

[0009] (a2) a nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 7 to 12, and optionally, wherein the nucleotide sequence is not naturally occurring;

[0010] (a3) the nucleotide sequence shown in any one of SEQ ID NOs: 13 to 18;

[0011] (a4) a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of any one of (a1) and (a3); wherein the nucleic acid molecule is capable of conferring resistance to a plant disease caused by at least one species of Phytophthora sp. on a plant comprising the nucleic acid molecule, and optionally wherein the nucleotide sequence is not naturally occurring;

[0012] (a5) A nucleotide sequence encoding an amino acid sequence having at least 75% sequence identity to any of the amino acid sequences shown in (a2), wherein the nucleic acid molecule is capable of conferring resistance to plant diseases caused by at least one subspecies of at least one species of the genus Phytophthora on a plant comprising the nucleic acid molecule, and optionally, wherein the nucleotide sequence is not naturally occurring.

[0013] Optionally, the nucleic acid molecule is a synthetic and / or isolated nucleic acid molecule.

[0014] Through extensive experiments, the present application discovered a wild diploid potato, Solanum cajamarquense, that is highly resistant to late blight. Whole-genome sequencing and evolutionary analysis were performed on this material, leading to the discovery of the candidate gene Rpi-caj1. The Rpi-caj1-C534 gene, first discovered in the present application, originated from the wild diploid potato, Solanum cajamarquense. Based on this gene sequence, the present application subsequently discovered Rpi-caj1-C813, Rpi-caj1-C550, Rpi-caj1-C509, Rpi-caj1-C450, and Rpi-caj1-C419 in Solanum cajamarquense, Solanum sogarandinum, Solanum cardiophyllum, Solanum piurae, and Solanum candolleanum. Different from many known late blight resistance genes, the amino acid sequences encoded by the above genes contain a TIR domain and a NB-ARC domain. This type of TNL gene was named Rpi-caj1 (or Rpi-cjm1).

[0015] In the present application, the amino acid sequence encoded by the above-mentioned Rpi-caj1 gene has three domains: TIR (Toll-like / Interleukin 1 receptor) domain: amino acids 8-185; NBS (nucleotide-binding site) domain: amino acids 203-433; LRR (eucine-rich repeats) domain: amino acids 433-606.

[0016] In the above (a4), the nucleotide sequences having at least 75% sequence identity are exemplified by nucleotide sequences having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 ...1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 Nucleotide sequences with 9.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity, preferably nucleotide sequences with 97.0%-98.0%, 97.5%-98.5%, 98.0%-99.0%, 98.5%-99.5%, 99.0%-100% identity.

[0017] In the above (a5), the nucleotide sequence encoding the amino acid sequence having at least 75% sequence identity is exemplified by a nucleotide sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1% or more. %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity, preferably a nucleotide sequence encoding an amino acid sequence having 97.0%-98.0%, 97.5%-98.5%, 98.0%-99.0%, 98.5%-99.5%, 99.0%-100% identity.

[0018] In some embodiments, the natural nucleotide sequence of the nucleic acid molecule related to the present application may be a variant sequence derived from different species and / or strains of Solanum, and its artificial nucleotide sequence (or artificial variant sequence) may be a variant sequence obtained by appropriate modification of the natural nucleotide sequence, and the modification includes but is not limited to appropriate nucleotide substitution / addition / deletion that does not affect the biological activity of the target protein, truncation of the N-terminal amino acid, codon optimization suitable for host cell preferences, addition of tags, fusion, etc., corresponding nucleotide variant sequences.

[0019] Preferably, the nucleotide sequence of Rpi-caj1 of the present application is at least one of the nucleotide sequences shown in (a1)-(a3) and artificial variant sequences thereof.

[0020] In a specific embodiment of the present application, the Rpi-caj1 nucleic acid molecule comprises at least one of the following nucleotide sequences:

[0021] (a1) the nucleotide sequence shown in SEQ ID NO: 1;

[0022] (a2) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 7;

[0023] (a3) the nucleotide sequence shown in SEQ ID NO: 13;

[0024] (a4) a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in any one of (a1) and (a3);

[0025] (a5) A nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with any of the amino acid sequences shown in (a2).

[0026] In a specific embodiment of the present application, in (a4), the nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 includes at least one of the nucleotide sequences shown in SEQ ID NOs: 2 to 6.

[0027] In a specific embodiment of the present application, in (a4), the nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 13 includes at least one of the nucleotide sequences shown in SEQ ID NOs: 14 to 18.

[0028] In a specific embodiment of the present application, in (a5), the nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 includes at least one of the nucleotide sequences shown in SEQ ID NOs: 8 to 12.

[0029] The Rpi-caj1 nucleotide sequences of the present application include but are not limited to the nucleotide sequences of wild-type Rpi-caj1-C534, Rpi-caj1-C813, Rpi-caj1-C550, Rpi-caj1-C509, Rpi-caj1-C450 and Rpi-caj1-C419 genes comprising a natural promoter and a 3' adjacent region containing the coding region, a cDNA sequence and a nucleotide sequence comprising only the coding region.

[0030] The present application also provides a nucleic acid molecule composition comprising the above nucleic acid molecule and an additional resistance (R) gene;

[0031] Optionally, the additional R genes include but are not limited to at least one of the following genes: Rpi-amr3i (Accession No. KT373889; SEQ ID NO: WO 2016 / 182881); NO:1), Rpi-blb1 (also known as "RB"; accession numbers FB764493.1 and AY336128.1), Rpi-sto1 (accession number EU884421), Rpi-pta1 (accession number EU884422), Rpi-blb2 (accession number DQ122125), Rpi-blb3 (accession number FJ536326), Rpi-abpt (accession number FJ536324), R2-like (accession number FJ536323), R2 (accession number FJ536325), Rpi-edn1.1 (accession number GU563963), Rpi-edn1.2, Rpi-snk1.1, Rpi-sn k1.2, Rpi-hjt1.1-Rpi-hjt1.3 (accession number GU563971-3), Rpi-bt1 (accession number FJ188415), R1 (accession number AF447489), R3a (accession number AY849382), R3b (accession number JF900492), Rpi-vnt1.1 (accession number FJ423044), Rpi-vnt1.2 (accession number FJ423045), Rpi-vnt1.3 (accession number FJ423046), Rpi-mcq1 (accession number GN043561), Rpi-chc, Ph-3 (accession number KJ563933) and R8 (accession number KU530153).

[0032] Nucleotide sequences corresponding to the accession numbers of the genes listed above, or any gene or protein disclosed elsewhere herein, can be obtained from publicly available online nucleotide and amino acid sequence databases, such as GenBank and the EMBL databases (available on the World Wide Web at ncbi.nlm.nih.gov / genbank and ebi.ac.uk, respectively).

[0033] The present application also provides a biomaterial, which is any one of the following (b1) to (b3):

[0034] (b1) an expression cassette or an expression cassette composition comprising the above-mentioned nucleic acid molecule or nucleic acid molecule composition;

[0035] (b2) a vector or vector composition comprising the aforementioned nucleic acid molecule or nucleic acid molecule composition, or a vector or vector composition comprising the expression cassette or expression cassette composition described in (b1);

[0036] (b3) A host cell containing the aforementioned nucleic acid molecule or nucleic acid molecule composition, or a host cell containing the expression cassette or expression cassette composition described in (b1), or a host cell containing the vector or vector composition described in (b2).

[0037] Optionally, the host cell comprises a bacterial or fungal cell.

[0038] In one embodiment of the present application, the biological material is an expression cassette or an expression cassette composition.

[0039] In the embodiments of the present application, when the nucleic acid molecule or nucleic acid molecule composition comprises at least two nucleic acid molecules, it can be prepared into one expression cassette or a composition consisting of at least two expression cassettes.

[0040] In an embodiment of the present application, the expression cassette or expression cassette composition further includes a regulatory element, and the regulatory element includes at least one of a promoter, an enhancer, a leader sequence, a transposon, a terminator, and a marker gene.

[0041] In an embodiment of the present application, the expression cassette or expression cassette composition further comprises an operably linked promoter.

[0042] Optionally, the promoter includes an operably linked endogenous promoter and / or an operably linked heterologous promoter.

[0043] Optionally, the operably linked endogenous promoter is the natural promoter of the Rpi-caj1 gene, such as the endogenous promoter Pcaj.

[0044] In an embodiment of the present application, the endogenous promoter Pcaj comprises at least one of the following nucleotide sequences: a nucleotide sequence as shown in any one of SEQ ID NOs: 19 to 24, and / or a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence as shown in any one of SEQ ID NOs: 19 to 24. Exemplary nucleotide sequences having at least 75% sequence identity are nucleotide sequences having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0045] In an embodiment of the present application, the endogenous promoter Pcaj comprises at least one of the following nucleotide sequences: the nucleotide sequence shown in SEQ ID NO: 19, and / or a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19.

[0046] In an embodiment of the present application, the nucleotide sequence having at least 75% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 includes at least one of the nucleotide sequences shown in SEQ ID NOs: 20 to 24.

[0047] In embodiments of the present application, the selection of an operably linked heterologous promoter may depend on many factors, such as desired timing, localization, and expression pattern, as well as responsiveness to specific biotic or abiotic stimuli. Optionally, the operably linked heterologous promoter includes at least one of a pathogen-inducible promoter, a constitutive promoter, a tissue-preferred promoter, a wound-inducible promoter, and a chemically regulated promoter. For example, the pblb3 promoter (Lokossou AA et al. (2009) Molecular plant-microbe interactions, 22(6):630-641.); the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J.3:2723-2730); ALS promoter (U.S. Patent No. 5,659,026), etc. Preferably, the promoter is suitable for stably expressing similar disease resistance genes in Solanum crops.

[0048] The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally should include 5' non-transcribed and 5' non-translated sequences for transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, and the like, as needed. In particular, such 5' non-transcribed regulatory sequences will include a promoter region, which includes a promoter sequence that controls transcriptional control of the operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as required. The expression cassettes of the present application may optionally include a 5' leader or signal sequence.

[0049] In an embodiment of the present application, the expression of a nucleic acid molecule can be regulated by manipulating the copy number of a gene or operon in a cell.

[0050] In some embodiments, the expression of nucleic acid molecules can be regulated by manipulating the order of nucleic acid molecules within a module.

[0051] In some embodiments, expression of a nucleic acid molecule is regulated by integrating one or more nucleic acid molecules or operons into a chromosome.

[0052] In another embodiment of the present application, the biological material is a carrier or a carrier composition.

[0053] In some embodiments, the biological material is a vector or vector composition containing the above-mentioned nucleic acid molecule or nucleic acid molecule composition.

[0054] In some embodiments, the biological material is a vector or vector composition containing the above-described expression cassette or expression cassette composition.

[0055] In some embodiments, one or more nucleic acid molecules of interest are expressed in an expression vector. As used herein, a "vector" can be any of a variety of nucleic acids into which one or more desired sequences can be inserted by restriction enzyme digestion and ligation for transport or expression in a host cell in a variety of genetic environments. Vectors are typically composed of DNA but can also be composed of RNA.

[0056] In an embodiment of the present application, the vector includes a plasmid, a chloroplast, a viral vector, a phage, a phagemid, a clay, a fosmid, a bacteriophage or an artificial chromosome; and optionally, the viral vector includes an adenoviral vector, a retroviral vector or an adeno-associated viral vector; and optionally, the vector includes a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC) or a mammalian artificial chromosome (MAC). For example, the vector includes pFastBac1, pYES2, pYES2.1, pESC-Ura, pESC-Trp, pESC-Leu, pESC-His, pGEX2T, pTAex3, pUSA, pYMB0, pHT43, pET28b, pIJ702, pUCP19, pYMB03, pHT43, pEAQ, pBin307, pPZP, pSAT, pCAMIA-1300, etc.

[0057] Cloning vectors are capable of autonomous replication or integration into the host cell genome and are further characterized by one or more restriction endonuclease sites at which the vector can be cut in a defined manner and a desired DNA sequence can be ligated into the vector so that the new plasmid retains its ability to replicate in the host cell. In the case where the vector is a plasmid, replication of the desired sequence can occur multiple times as the copy number of the plasmid increases in the host cell (e.g., a bacterial host), or only once per host before the host reproduces by mitosis. In the case where the vector is a bacteriophage, replication can occur actively during the lytic phase or passively during the lysogenic phase.

[0058] Expression vectors can be inserted into the desired DNA sequence by restriction enzyme digestion and connection so that the desired DNA sequence is effectively connected to the regulatory sequence and can be expressed as an RNA transcript. The vector can also contain one or more marker sequences suitable for identifying whether the cell has been transformed or transfected by the vector. Markers include genes that increase or decrease the protein of its resistance or sensitivity to antibiotics or other compounds, genes (such as beta-galactosidase, luciferase or alkaline phosphatase) that encode enzymes that can detect activity by standard methods known in the art, and genes (such as green fluorescent protein) that have a visible effect on the phenotype of transformed or transfected cells, hosts, bacterium colonies or plaques. Preferred vectors are vectors that can autonomously replicate and express the structural gene products present in the DNA fragments that are effectively connected thereto.

[0059] In the embodiments of the present application, when the nucleic acid molecule or nucleic acid molecule composition includes at least two nucleic acid molecules, it can be prepared into one vector or a composition consisting of at least two vectors.

[0060] In another embodiment of the present application, the biological material is a host cell.

[0061] In some embodiments, the biological material is a host cell containing the above-mentioned nucleic acid molecule or nucleic acid molecule composition.

[0062] In some embodiments, the biological material is a host cell for the expression cassette or expression cassette composition described above.

[0063] In some embodiments, the biological material is a host cell of the above-described vector or vector composition.

[0064] Optionally, the host cell comprises a bacterial or fungal cell.

[0065] The present application also provides a promoter for driving transcription of the Rpi-caj1 gene, wherein the promoter Pcaj comprises at least one of the following nucleotide sequences: a nucleotide sequence as shown in any one of SEQ ID NOs: 19 to 24, and / or a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence as shown in any one of SEQ ID NOs: 19 to 24. Exemplary nucleotide sequences having at least 75% sequence identity are nucleotide sequences having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0066] In an embodiment of the present application, the endogenous promoter Pcaj comprises at least one of the following nucleotide sequences: the nucleotide sequence shown in SEQ ID NO: 19, and / or a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19.

[0067] In an embodiment of the present application, the nucleotide sequence having at least 75% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 includes at least one of the nucleotide sequences shown in SEQ ID NOs: 20 to 24.

[0068] The present application provides a method for producing a host cell, which comprises transforming the host cell with at least one of the above-mentioned nucleic acid molecule or nucleic acid molecule composition, expression cassette or expression cassette composition, vector or vector composition.

[0069] The present application provides the use of any one of the nucleic acid molecules, nucleic acid molecule compositions, and biological materials of the present application in enhancing a plant's resistance to a plant disease caused by at least one subspecies of at least one Phytophthora species.

[0070] In an embodiment of the present application, the plant includes a plant of the Solanaceae family.

[0071] Optionally, the Solanaceae plant includes at least one of potato, tomato, eggplant, pepper, tobacco, petunia, tomatillo, and cape gooseberry.

[0072] Preferably, the plant of the Solanaceae family is potato and / or tomato.

[0073] The present application also provides a method for enhancing a plant's resistance to a plant disease caused by at least one subspecies of at least one Phytophthora species, the method comprising modifying at least one plant cell to contain a heterologous polynucleotide comprising the above-mentioned nucleic acid molecule or nucleic acid molecule composition.

[0074] In a preferred embodiment of the present application, the species of Phytophthora is Phytophthora infestans. In other embodiments, the species of Phytophthora is a species of Phytophthora that can cause plant diseases on at least one plant. For the present application, some species of Phytophthora include but are not limited to Phytophthora infestans, Phytophthora parasitica, Phytophthora ramorum, Phytophthora ipomoeae, Phytophthora mirabilis, Phytophthora capsici, Phytophthora porri, Phytophthora sojae, Phytophthora palmivora, and Phytophthora phaseoli.

[0075] In an embodiment of the present application, a plant comprising the heterologous polynucleotide has enhanced resistance to a plant disease caused by at least one race of at least one Phytophthora species relative to a control plant (a control plant not comprising the heterologous polynucleotide).

[0076] In an embodiment of the present application, modifying at least one plant cell to contain a heterologous polynucleotide specifically comprises: transforming at least one of the nucleic acid molecules, nucleic acid molecule compositions, or expression cassettes or expression cassette compositions, vectors or vector compositions in biological materials of the present application into plant cells, so that the plant cells express the protein.

[0077] In the above methods, methods for transforming nucleic acid molecules, nucleic acid molecule compositions, expression cassettes, and vectors into plant cells include but are not limited to Agrobacterium-mediated transformation, gene gun transformation, electroporation, polyethylene glycol (PEG) transformation, lipid transfection, heat shock, calcium phosphate precipitation, virus-mediated, microinjection, and genetic engineering editing technology.

[0078] In a specific embodiment of the present application, the method for expressing a protein is, for example: (1) constructing a vector comprising the nucleic acid molecule or nucleic acid molecule composition of the present application; (2) transforming the obtained vector into a plant cell; and (3) culturing the obtained plant cell to express the gene to produce a protein.

[0079] In an embodiment of the present application, the method for enhancing plant resistance further comprises regenerating the plant cell into a plant comprising the heterologous polynucleotide in its genome.

[0080] Preferably, the regenerated plants comprise enhanced resistance to a plant disease caused by at least one race of at least one species of Phytophthora relative to the resistance of control plants to said plant disease.

[0081] Preferably, plants comprising the heterologous polynucleotide have enhanced resistance to plant diseases caused by at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or more races of a species of the genus Phytophthora relative to control plants.

[0082] In an embodiment of the present application, the resistance of a plant comprising the heterologous polynucleotide in its genome to a plant disease caused by at least one species of at least one Phytophthora is enhanced or increased by at least 25% compared to a control plant, for example, by at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or more.

[0083] In some embodiments, the plant disease caused by at least one race of at least one Phytophthora species is late blight.

[0084] Depending on the desired outcome, the heterologous polynucleotide of the present application can be stably integrated into the genome of the plant cell or stably integrated into the genome of the plant cell.

[0085] For example, if the desired result is to produce a stably transformed plant with enhanced resistance to a plant disease caused by at least one species of a certain species of Phytophthora, the heterologous polynucleotide can be, for example, fused to a plant transformation vector suitable for stably integrating the heterologous polynucleotide into the plant cell genome. In some embodiments, the nucleic acid molecule or nucleic acid molecule composition (heterologous polynucleotide) of the present application can be stably integrated into the genome of the host cell. Such stably transformed plants can pass the heterologous polynucleotide to subsequent generations of offspring plants by sexual reproduction and / or asexual reproduction.

[0086] In an embodiment of the present application, the modifying of at least one plant cell to include a heterologous polynucleotide comprises genome editing technology.

[0087] In other embodiments herein, in which stable integration of the heterologous polynucleotide into the plant genome is undesirable, transient transformation methods can be used to introduce the heterologous polynucleotide into one or more plant cells of the plant. In other embodiments, the heterologous polynucleotide is stably integrated into the genome of the host cell by microinjection, microparticle bombardment, viral vector infection, or by applying a modified virus and / or modified viral nucleic acid to the plant or part thereof by spraying, irrigation, or dusting, thereby causing the host or host cell to transiently express the gene of interest.

[0088] In the embodiments of the present application, the plant includes any plant species, such as monocots, dicots, and conifers.

[0089] In a specific embodiment, the plants of the present application include crop plants, such as corn, soybean, wheat, rice, cotton, alfalfa, sunflower, Brassica (certain species of the genus Brassica, particularly Brassica napus, Brassica rapa, Brassica juncea), rapeseed (Brassica napus), sorghum, millet, barley, triticale, safflower, peanut, sugarcane, tobacco, potato, tomato, eggplant and pepper.

[0090] Preferably, the plant comprises a plant of the Solanaceae family.

[0091] Preferably, the Solanaceae plant includes but is not limited to at least one of potato, tomato, eggplant, pepper, tobacco, petunia, tomatillo, and cape gooseberry.

[0092] In a more preferred embodiment, the Solanaceae plant is potato and / or tomato.

[0093] The present application also provides a method for controlling plant diseases caused by at least one species of at least one Phytophthora species in crop production, the method comprising:

[0094] modifying at least one plant cell to contain a heterologous polynucleotide comprising the nucleic acid molecule or nucleic acid molecule combination described above;

[0095] regenerating the plant cell into a plant comprising the heterologous polynucleotide in its genome;

[0096] The seedlings, tubers or seeds of the plants are planted and cultivated under conditions conducive to the growth and development of the plants.

[0097] In an embodiment of the present application, the method further comprises harvesting at least one of fruits, tubers, leaves, and seeds from the plant.

[0098] The present application also provides a method for detecting plant disease resistance, which comprises detecting the presence of the above-mentioned nucleic acid molecule or nucleic acid molecule composition in a plant, a plant part thereof or a plant cell.

[0099] In an embodiment of the present application, the plant disease resistance comprises resistance to a plant disease caused by at least one race of at least one Phytophthora species.

[0100] In some embodiments, the plant disease caused by at least one race of at least one Phytophthora species is late blight.

[0101] In some embodiments, the method for detecting plant disease resistance can be used to breed Solanaceae plants that are resistant to plant diseases caused by certain species of Phytophthora (e.g., late blight). Such resistant plants can be used for agricultural production of fruits, tubers, leaves and / or seeds for human or animal consumption or other uses.

[0102] In an embodiment of the present application, the method for detecting the presence of the nucleic acid molecule or nucleic acid molecule composition includes: detecting the presence of the nucleic acid molecule or nucleic acid molecule composition by detecting the complete nucleic acid molecule or nucleic acid molecule composition, or detecting at least one molecular marker in the nucleic acid molecule or nucleic acid molecule composition.

[0103] In an embodiment of the present application, the method for detecting the presence of the above-mentioned nucleic acid molecule or nucleic acid molecule composition includes PCR amplification, nucleic acid sequencing, nucleic acid hybridization or immunological assay for detecting the protein or polypeptide encoded by the nucleic acid molecule or nucleic acid molecule composition.

[0104] In some embodiments, in a method for detecting a Solanaceae plant that exhibits newly conferred or enhanced resistance to a plant disease caused by at least one subspecies of a species of the genus Phytophthora, detecting the presence of an Rpi-caj1 nucleotide sequence in the Solanaceae plant can involve one or more of the following molecular biology techniques known in the art, including but not limited to isolating genomic DNA and / or RNA from the plant, amplifying a nucleic acid molecule comprising an Rpi-caj1 nucleotide sequence and / or a molecular marker herein by PCR amplification, sequencing a nucleic acid molecule comprising an Rpi-caj1 nucleotide sequence and / or a molecular marker, detecting an Rpi-caj1 nucleotide sequence, a molecular marker, or a transcript of an Rpi-caj1 nucleotide sequence by nucleic acid hybridization, and performing an immunological assay for detecting an R protein encoded by an Rpi-caj1 nucleotide sequence. Specifically, oligonucleotide probes and PCR primers can be designed to detect the Rpi-caj1 nucleotide sequence of the present application, and such probes and PCR primers can be used in methods disclosed elsewhere herein or known in the art to rapidly detect in a plant population one or more plants comprising the Rpi-caj1 nucleotide sequence of the present application.

[0105] In other embodiments of the present application, detecting the presence of the Rpi-caj1 nucleotide sequence comprises detecting the presence of the R protein encoded by the Rpi-caj1 nucleotide sequence using, for example, an immunological detection method involving antibodies specific for the R protein.

[0106] The present application also provides a method for selecting plants, which comprises: detecting the presence of the above-mentioned nucleic acid molecules or nucleic acid molecule compositions in plants, plant parts thereof or plant cells thereof; and selecting plants containing at least one copy of the nucleic acid molecules or nucleic acid molecule compositions in their genomes.

[0107] In an embodiment of the present application, the method for detecting the presence of the nucleic acid molecule or nucleic acid molecule composition includes: detecting the presence of the nucleic acid molecule or nucleic acid molecule composition by detecting the complete nucleic acid molecule or nucleic acid molecule composition, or detecting at least one molecular marker in the nucleic acid molecule or nucleic acid molecule composition.

[0108] In an embodiment of the present application, the method for detecting the presence of the above-mentioned nucleic acid molecule or nucleic acid molecule composition includes: PCR amplification, nucleic acid sequencing, nucleic acid hybridization or immunological assay for detecting the protein or polypeptide encoded by the nucleic acid molecule or nucleic acid molecule composition.

[0109] In some embodiments, in a method for detecting a Solanaceae plant that exhibits newly conferred or enhanced resistance to a plant disease caused by at least one subspecies of a species of the genus Phytophthora, detecting the presence of an Rpi-caj1 nucleotide sequence in the Solanaceae plant may involve one or more of the following molecular biology techniques known in the art, including but not limited to: isolating genomic DNA and / or RNA from the plant, amplifying a nucleic acid molecule comprising an Rpi-caj1 nucleotide sequence and / or a molecular marker herein by PCR amplification, sequencing a nucleic acid molecule comprising an Rpi-caj1 nucleotide sequence and / or a molecular marker, detecting an Rpi-caj1 nucleotide sequence, a molecular marker, or a transcript of an Rpi-caj1 nucleotide sequence by nucleic acid hybridization, and performing an immunological assay for detecting an R protein encoded by an Rpi-caj1 nucleotide sequence. Specifically, oligonucleotide probes and PCR primers can be designed to detect the Rpi-caj1 nucleotide sequence of the present application, and such probes and PCR primers can be used in methods disclosed elsewhere herein or known in the art to rapidly detect one or more plants comprising the presence of an Rpi-caj1 nucleotide sequence of the present application in a plant population.

[0110] In an embodiment of the present application, detecting the presence of the nucleic acid molecule or nucleic acid molecule composition can be achieved by using primers shown in SEQ ID NOs: 25-38.

[0111] In other embodiments of the present application, detecting the presence of the Rpi-caj1 nucleotide sequence comprises detecting the presence of the R protein encoded by the Rpi-caj1 nucleotide sequence using, for example, an immunological detection method involving antibodies specific for the R protein.

[0112] The present application also provides a method for introducing the above nucleic acid molecule or nucleic acid molecule composition into a plant, the method comprising:

[0113] (c1) hybridizing the first plant with the second plant to produce offspring plants;

[0114] The first plant is a plant comprising at least one copy of the above-mentioned nucleic acid molecule or nucleic acid molecule composition in its genome;

[0115] The second plant is a plant lacking the nucleic acid molecule or nucleic acid molecule combination in its genome;

[0116] (c2) selecting an offspring plant comprising in its genome at least one copy of the nucleic acid molecule or the combination of nucleic acid molecules.

[0117] In the embodiment of the present application, (c2) is specifically:

[0118] detecting the presence of the nucleic acid molecule or nucleic acid molecule combination described above in progeny plants, plant parts thereof, or plant cells thereof;

[0119] Progeny plants are selected that comprise in their genome at least one copy of the nucleic acid molecule or combination of nucleic acid molecules.

[0120] In the present application embodiment, the first plant and the second plant can be same species or can be different species.This hybridization of the first species of plant and the second species of plant is called interspecific hybridization, and can be used for one or more target genes being infiltrated into the related species that lacks these one or more target genes from a species, and generally relate to the multi-generation backcross of offspring and related species and select the offspring that comprises one or more target genes in each generation.This interspecific hybridization, gene infiltration and backcross method are well known in the art, and can be used for the method for the application.

[0121] In the method for introducing at least one Rpi-caj1 gene of the present application into a plant that lacks the at least one Rpi-caj1 gene in its genome, the first plant or the second plant can be a pollen donor plant. For example, if the first plant is a pollen donor plant, the second plant is a pollen recipient plant. Similarly, if the second plant is a pollen donor plant, the first plant is a pollen recipient plant. After hybridization, the pollen recipient plant is grown under conditions that are conducive to plant growth and development, and grown for a sufficient time to allow the seeds to mature or achieve other desired growth stages for subsequent in vitro germination processes (such as embryo rescue). The seeds can then be harvested and those seeds containing the Rpi-caj1 gene can be detected by any method known in the art, including, for example, methods described elsewhere herein for detecting Solanaceae plants that show newly conferred or enhanced resistance to plant diseases caused by at least one subspecies of a species of the genus Phytophthora. In certain embodiments, the first plant is a potato plant comprising one or more Rpi-caj1 genes and the second plant is a potato plant lacking the one or more Rpi-caj1 genes.

[0122] The present application also provides a plant or a plant part thereof, wherein the plant is one of the following plants:

[0123] (d1) a plant comprising at least one of the above nucleic acid molecules, nucleic acid molecule compositions, expression cassettes, vectors or host cells;

[0124] (d2) plants grown from plant cells modified to contain the aforementioned nucleic acid molecules or nucleic acid molecule compositions;

[0125] (d3) producing a plant obtained by the method for increasing the resistance of a plant to a plant disease caused by at least one species of at least one Phytophthora;

[0126] (d4) progeny formed by self-pollination of any one of the plants in (d1) to (d3), and plants grown from the progeny;

[0127] (d5) Offspring formed by hybridizing any plant among (d1)-(d3) with other varieties, and plants grown from the offspring; preferably, the hybridization adopts one of the above-mentioned methods for introducing the above-mentioned nucleic acid molecules or nucleic acid molecule compositions into plants.

[0128] The plant parts mentioned above are roots, stems, tubers, leaves, flowers, fruits, pollen or seeds.

[0129] The present application provides plants comprising nucleic acid molecules or nucleic acid molecule compositions (heterologous polynucleotides), wherein the heterologous polynucleotide comprises an R gene nucleotide sequence of the present application. Preferably, the R gene nucleotide sequence encodes the full-length R protein of the present application, or at least one or more functional portions or one or more domains thereof.

[0130] The plants disclosed herein are useful for controlling plant diseases caused by at least one race of at least one Phytophthora species in agricultural crop production, particularly in areas where such plant diseases are prevalent and known to negatively impact, or at least potentially negatively impact, agricultural yields.

[0131] In a specific embodiment, the plants of the present application include crop plants, such as corn, soybean, wheat, rice, cotton, alfalfa, sunflower, Brassica (certain species of the genus Brassica, particularly Brassica napus, Brassica rapa, Brassica juncea), rapeseed (Brassica napus), sorghum, millet, barley, triticale, safflower, peanut, sugarcane, tobacco, potato, tomato, eggplant and pepper.

[0132] Preferably, the plant comprises a plant of the Solanaceae family.

[0133] Preferably, the Solanaceae plant includes but is not limited to at least one of potato, tomato, eggplant, pepper, tobacco, petunia, tomatillo, and cape gooseberry.

[0134] In a more preferred embodiment, the Solanaceae plant is potato and / or tomato.

[0135] The present application also provides a plant or a plant part thereof, wherein the plant includes a transgenic plant containing the above-mentioned nucleic acid molecule or nucleic acid molecule composition.

[0136] In a specific embodiment, the plants or plant parts thereof of the present application include crop plants, such as corn, soybean, wheat, rice, cotton, alfalfa, sunflower, Brassica (certain species of the genus Brassica, particularly Brassica napus, Brassica rapa, Brassica juncea), rapeseed (Brassica napus), sorghum, millet, barley, triticale, safflower, peanut, sugarcane, tobacco, potato, tomato, eggplant and pepper.

[0137] In a specific embodiment, the plant or plant part thereof comprises a plant of the Solanaceae family, which includes but is not limited to at least one of potato, tomato, eggplant, pepper, tobacco, petunia, tomatillo, and cape gooseberry.

[0138] In a more preferred embodiment, the Solanaceae plant is potato and / or tomato.

[0139] The present application also provides a transgenic plant or a plant part thereof, wherein the plant comprises the above-mentioned nucleic acid molecule or nucleic acid molecule composition, and a promoter, wherein the above-mentioned nucleic acid molecule or nucleic acid molecule composition is operably linked to the above-mentioned promoter.

[0140] Preferably, the promoter includes an operably linked endogenous promoter and / or an operably linked heterologous promoter.

[0141] The present application also provides a method for producing a plant, which comprises transforming the plant with at least one of the above-mentioned nucleic acid molecule or nucleic acid molecule composition, expression cassette or expression cassette composition, vector or vector composition.

[0142] The present application also provides a plant prepared by the above method for producing a plant.

[0143] In a specific embodiment, the plants of the present application include crop plants, such as corn, soybean, wheat, rice, cotton, alfalfa, sunflower, Brassica (certain species of the genus Brassica, particularly Brassica napus, Brassica rapa, Brassica juncea), rapeseed (Brassica napus), sorghum, millet, barley, triticale, safflower, peanut, sugarcane, tobacco, potato, tomato, eggplant and pepper.

[0144] Preferably, the plant comprises a plant of the Solanaceae family.

[0145] Preferably, the Solanaceae plant includes but is not limited to at least one of potato, tomato, eggplant, pepper, tobacco, petunia, tomatillo, and cape gooseberry.

[0146] In a more preferred embodiment, the Solanaceae plant is potato and / or tomato.

[0147] The present application also provides an agricultural product for human or animal consumption, which includes the above-mentioned plant or its plant part, or the agricultural product for human or animal consumption is a product made from the above-mentioned plant or its plant part.

[0148] In the embodiment of the present application, the agricultural product for human or animal consumption comprises food and other agricultural products.Other agricultural products comprise the tobacco products (such as cigarettes, cigars, pipes and chewing tobacco) produced from tobacco leaves and the food and industrial starch products produced from potato tubers.Such food can be consumed or used by people and other animals, and the animal includes but is not limited to pets (such as dogs and cats), livestock (such as pigs, cattle, chickens, turkeys and ducks) and the animals (such as fish, shrimps, prawns, crayfish and lobsters) produced in freshwater and seawater aquaculture systems.

[0149] The present application also provides an Rpi-caj1 protein comprising at least one of the following amino acid sequences:

[0150] (e1) the amino acid sequence shown in any one of SEQ ID NOs: 7 to 12;

[0151] (e2) an amino acid sequence encoded by the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 6;

[0152] (e3) an amino acid sequence encoded by the nucleotide sequence shown in any one of SEQ ID NOs: 13 to 18;

[0153] (e4) an amino acid sequence having at least 75% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 7 to 12, wherein a protein comprising the amino acid sequence is capable of conferring resistance to plant diseases caused by at least one subspecies of at least one species of the genus Phytophthora on a plant comprising the protein.

[0154] In the above (e4), the amino acid sequences having at least 75% sequence identity are exemplified by amino acid sequences having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 ...1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 amino acid sequences with 9.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity, preferably amino acid sequences with 97.0%-98.0%, 97.5%-98.5%, 98.0%-99.0%, 98.5%-99.5%, 99.0%-100% identity.

[0155] In a specific embodiment of the present application, an Rpi-caj1 protein comprises at least one of the following amino acid sequences:

[0156] (e1) the amino acid sequence shown in SEQ ID NO:7;

[0157] (e2) an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1;

[0158] (e3) an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 13 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 13;

[0159] (e4) an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 7, wherein a protein comprising the amino acid sequence is capable of conferring resistance to a plant disease caused by at least one race of at least one species of the genus Phytophthora on a plant comprising the protein;

[0160] (e5) a fusion amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 7;

[0161] (e6) An amino acid sequence having the same function as that of SEQ ID NO: 7 obtained by substitution and / or deletion and / or addition of one or more amino acid residues.

[0162] In a specific embodiment of the present application, in (e2), the nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 includes at least one of the nucleotide sequences shown in SEQ ID NOs: 2 to 6.

[0163] In a specific embodiment of the present application, the nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 13 includes at least one of the nucleotide sequences shown in SEQ ID NO: 14 to 18;

[0164] In a specific embodiment of the present application, the amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 includes at least one of the nucleotide sequences shown in SEQ ID NOs: 8 to 12.

[0165] In some embodiments, the natural amino acid sequence of the aforementioned protein may be a variant sequence derived from different species and / or strains of the genus Solanum. The artificial variant amino acid sequence of the aforementioned protein may be a variant sequence obtained by appropriate modification of the natural amino acid sequence, including but not limited to appropriate amino acid substitutions / additions / deletions that do not affect the biological activity of the target protein, truncation of the N-terminal amino acid, codon optimization suitable for host cell preference, addition of tags, fusion, etc.

[0166] Preferably, the amino acid sequence of Rpi-caj1 of the present application is at least one of the amino acid sequences shown in (e1)-(e3) and artificial variant sequences thereof.

[0167] Compared with the prior art, the present invention has the following beneficial effects:

[0168] The Rpi-caj1 gene of the present application can confer broad-spectrum resistance to late blight infection caused by Phytophthora infestans on potatoes, for example, it can simultaneously produce significant resistance to 16 different strains of Phytophthora infestans.

[0169] The present application discloses that transient expression of the Rpi-caj1 gene in Nicotiana benthamiana leaves can confer resistance to late blight on Nicotiana benthamiana leaves.

[0170] This application provides important genetic resources for breeding potato varieties resistant to late blight, overcomes the problem of loss of disease resistance in potato varieties, and provides new guarantees for the long-term disease resistance of potatoes. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Figure 1. Effect of transient expression of Rpi-caj1-C534 in Nicotiana benthamiana leaves on inoculation with Phytophthora infestans. The left side of the leaf shows transient expression of Rpi-caj1-C534 (CaMV35S:Rpi-caj1 construct), while the right side of the leaf shows the control (CaMV35S:gfp). The blue area in the color image shows the lesion (the black area in the black and white image shows the lesion). dpi is the abbreviation for days post inoculation, and 5 dpi means 5 days post inoculation. **** indicates a P value less than 0.0001.

[0172] Figure 2 Effects of transient expression of Rpi-caj1 homologous genes from different sources in tobacco leaves on inoculation with Phytophthora infestans; **** indicates a P value less than 0.0001;

[0173] Figure 3 is an electrophoretic diagram of Rpi-caj1-C534 transformed into the potato genome; numbers 1-22 indicate the strain numbers of Rpi-caj1-C534 successfully transformed into the potato genome, control 1 (negative control) is the amplification result of the untransformed potato genome, and control 2 (positive control) is the amplification result of the genome of Solanum cajamarquense material naturally containing the Rpi-caj1-C534 gene; * indicates the position of the target amplified band;

[0174] Figure 4 shows the inoculation effect of Rpi-caj1-C534 transgenic potato leaves on Phytophthora infestans; strains #3, #4, and #13 are the positive potato transformation lines transformed with the Rpi-caj1-C534 gene verified by PCR in Figure 3. They are three independent strains obtained after transformation with the same gene and are used to verify Rpi-caj1-C534-mediated late blight resistance.

[0175] The English annotations in the figure are as follows:

[0176] Infection area: infection area;

[0177] Rpi-blb2: Désirée potato variety with the Rpi-blb2 gene. DETAILED DESCRIPTION

[0178] This application discloses late blight resistance genes, biomaterials, and applications. Those skilled in the art can draw upon the content herein and appropriately modify process parameters to achieve these results. It is particularly important to note that all similar substitutions and modifications readily apparent to those skilled in the art are considered encompassed by this application. The methods and applications of this application have been described using preferred embodiments. It is apparent that those skilled in the art can modify, adapt, and combine the methods and applications described herein to implement and apply the technology of this application without departing from the content, spirit, and scope of this application.

[0179] Explanation of terms:

[0180] As used herein, the term "nucleic acid molecule" (or "nucleic acid" or "polynucleotide") may refer to a polymeric form of nucleotides, which may include sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers of the above. Nucleotides may refer to ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide." A nucleic acid molecule is typically at least 10 bases in length, unless otherwise indicated. The term may refer to RNA or DNA molecules of indefinite length. The term includes single-stranded and double-stranded forms of DNA. Nucleic acid molecules may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotides. The present application provides nucleic acid molecules comprising the nucleotide sequence of an Rpi-caj1 gene (particularly Rpi-caj1-C534, Rpi-caj1-C813, Rpi-caj1-C550, Rpi-caj1-C509, Rpi-caj1-C450, Rpi-caj1-C419 and alleles, homologs, orthologs and other naturally occurring variants of such R genes, as well as synthetic or artificial (i.e., non-naturally occurring) variants thereof). Therefore, the genes and polynucleotides of the present application include naturally occurring sequences as well as mutants and other variant forms.

[0181] The term "variant" is intended to mean a substantially similar sequence. For polynucleotides, variants are included in polynucleotides having a deletion (i.e., truncation) at the 5' and / or 3' ends; and / or having a deletion and / or addition of one or more nucleotides at one or more internal sites in a natural polynucleotide; and / or having a polynucleotide substituted with one or more nucleotides at one or more sites in a natural polynucleotide. As used herein, "natural" polynucleotides or polypeptides comprise naturally occurring nucleotide sequences or amino acid sequences, respectively. For polynucleotides, conservative variants include those sequences that encode the amino acid sequence of one of the R proteins of the present application due to the degeneracy of the genetic code. Naturally occurring allelic variants can be identified using well-known molecular biology techniques (e.g., polymerase chain reaction (PCR) and hybridization techniques). Variant polynucleotides also include synthetically derived polynucleotides, such as those produced by using site-directed mutagenesis but still encoding the R protein of the present application. Typically, as determined by sequence alignment programs and parameters, variants of the specific polynucleotides of the present application will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the specific polynucleotide. In certain embodiments of the present application, variants of the specific polynucleotides of the present application will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with at least one nucleotide sequence selected from SEQ ID NOs: 1 to 6, and optionally comprise a non-naturally occurring nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NOs: 1 to 6 by at least one nucleotide modification selected from the group consisting of substitution of at least one nucleotide, addition of at least one nucleotide, and deletion of at least one nucleotide. It should be understood that the addition of at least one nucleotide can be the addition of one or more nucleotides within the nucleotide sequence of the present application, the addition of one or more nucleotides to the 5' end of the nucleotide sequence of the present application, and / or the addition of one or more nucleotides to the 3' end of the nucleotide sequence of the present application.

[0182] As will be readily appreciated by those skilled in the art, nucleic acid molecules can be chemically or biochemically modified, or can comprise non-natural or derived nucleotide bases, such modifications including, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications (e.g., uncharged bonds: such as methyl phosphonates, phosphotriesters, phosphoramidites, carbamates, etc.; charged bonds: such as phosphorothioates, phosphorodithioates, etc.; pendant moieties: such as peptides; intercalators: such as acridine, psoralen, etc.; chelators; alkylating agents; and modified bonds: such as α-anomeric nucleic acids, etc.), and the term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpin, circular, and padlocked conformations.

[0183] As those skilled in the art will readily appreciate, naturally occurring allelic variants can be identified through hybridization techniques under stringent conditions. "Stringent conditions" can be any of low, moderate, or high stringency conditions. "Low stringency conditions" include, for example, 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Moderate stringency conditions" include, for example, 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "High stringency conditions" include, for example, 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Among these conditions, increasing the temperature increases the likelihood of obtaining DNA with high homology. Hybridization stringency can be influenced by various factors, including temperature, probe concentration, probe length, ionic strength, time, and salt concentration. By appropriately selecting these factors, those skilled in the art can achieve similar stringency conditions.

[0184] In this application, the term "identity" refers to sequence similarity to an exemplary nucleic acid sequence or amino acid sequence. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0185] The sequence identity values ​​provided herein refer to the full-length sequence identity evaluation of the sequence using the Dispatcher tools framework (https: / / www.ebi.ac.uk / services). Other software can also be used to obtain sequence identity values, such as the values ​​obtained by using the default parameters in the multiple alignment software package MUSCLE v3.8.31 ("MUSCLE: multiple sequence alignment with high accuracy and high throughput" Nucleic Acids Res. 32 (5): 1792 (2004)) using Jalview version 2.11.2.7 (Waterhouse, AM, Procter, JB, Martin, DMA, Clamp, M. and Barton, GJ (2009) "Jalview Version 2-a multiple sequence alignment editor and analysis workbench" Bioinformatics 25 (9) 1189-1191 doi: 10.1093 / bioinformatics / btp033); or any equivalent thereof.

[0186] Other mathematical algorithms are known in the art and can be used to align two sequences. See, for example, the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm was incorporated into the BLAST program of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using the BLASTN program (nucleotide query for nucleotide sequence searches) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application, or using the BLASTX program (translated nucleotide query for protein sequence searches) to obtain protein sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed with the BLASTP program (protein query for protein sequence searches) to obtain amino acid sequences homologous to the protein molecules of the present application, or with the TBLASTN program (protein query for translated nucleotide sequence searches) to obtain nucleotide sequences homologous to the protein molecules of the present application. In order to obtain gap alignments for comparison purposes, Gapped BLAST (in BLAST2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterative search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the corresponding programs (e.g., BLASTX and BLASTN) can be used. Alignment can also be performed manually by inspection.

[0187] In this application, the term "homology" is sometimes used to refer to the level of similarity between two or more nucleic acid or amino acid sequences (i.e., sequence similarity or identity) expressed as a percentage of positional identity. Homology also refers to the concept of evolutionary relatedness, typically demonstrated by similar functional properties between different nucleic acids or proteins that share similar sequences.

[0188] In some embodiments, homologous sequences related to the present application can be obtained by comparing the exemplary sequences with the genome or transcriptome data of samples of evolutionarily closely related species. For example, between different species of the same genus or between different strains of the same species, by comparing the exemplary sequences with the genome or transcriptome data of the samples, a person skilled in the art can expect that the homologous sequences of the sequences will have the same or similar functions.

[0189] As used herein, the term "CDS" refers to a coding sequence. DNA is transcribed into mRNA, which is then translated into protein through splicing and other processing. A CDS is a DNA sequence that corresponds one-to-one with the protein sequence, without any other sequences not corresponding to the protein. Regardless of sequence changes during mRNA processing, the CDS completely corresponds to the protein's codons.

[0190] In some embodiments, the nucleic acid molecules associated with the present application can be cloned from DNA comprising a given nucleic acid molecule from any source, for example, by PCR amplification and / or restriction enzyme digestion. In some embodiments, the nucleic acid molecules associated with the present application are synthetic. Any method for obtaining the nucleic acid molecules associated with the invention is compatible with the present application.

[0191] In this application, the term "synthetic" refers to polynucleotide (i.e., DNA or RNA) molecules produced by chemical synthesis as an in vitro process. For example, TM Synthetic DNA is produced during the reaction process in the tube so that the synthetic DNA is enzymatically produced from natural DNA or RNA chains. Other laboratory methods can be used to synthesize polynucleotide sequences. Oligonucleotides can be chemically synthesized on an oligonucleotide synthesizer using solid phase synthesis using phosphoramidites. The synthesized oligonucleotides can be annealed to each other as complexes to produce "synthetic" polynucleotides. Other methods for chemically synthesizing polynucleotides are known in the art and can be easily implemented for use in the present disclosure.

[0192] As used herein, the term "gene" refers to a nucleic acid fragment that expresses a specific protein. A "gene" includes the DNA region that encodes the gene product, as well as all DNA regions that regulate the production of the gene product, regardless of whether such regulatory sequences are adjacent to the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, introns, and locus control regions.

[0193] As used herein, the term "gene product" refers to any product produced by a gene. For example, a gene product can be a direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA, ribozyme, structural RNA, or any other type of RNA), or a protein produced by translation of an mRNA.

[0194] In this application, the term "protein" (or "protein" or "peptide" or "polypeptide" or "peptide composition") includes both naturally occurring proteins and variants and modified forms thereof. As used herein, the terms "protein" and "polypeptide" are used interchangeably, so the term polypeptide can be used to refer to a full-length polypeptide or a fragment of a full-length polypeptide. The term "fragment" refers to a portion of a polypeptide sequence. "Fragments" or "biologically active portions" include polypeptides that contain a sufficient number of contiguous amino acid residues to retain biological activity, such as polypeptides in which the N-terminal amino acid is truncated.

[0195] "Variant" protein refers to a protein derived from a natural protein by the deletion of one or more amino acids at the N-terminus and / or C-terminus of the natural protein (so-called truncation), the deletion and / or addition of one or more amino acids at one or more internal sites in the natural protein, or the substitution of one or more amino acids at one or more sites in the natural protein. Such variants can be produced, for example, by genetic polymorphism or artificial manipulation. As determined by sequence alignment programs and parameters, biologically active variants of R proteins will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the amino acid sequences of the present application (e.g., the amino acid sequences shown in SEQ ID NOs: 7 to 12). The biologically active variants of the protein of the present application may differ from the protein in as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2 or even 1 amino acid residue. More preferably, such variants confer enhanced resistance to plant diseases caused by at least one species of at least one Phytophthora species on the plant or part thereof comprising the variant. In some embodiments, the mutations to be created in the DNA encoding the variant will not place the sequence out of reading frame. Optimally, the mutations will not create complementary regions that could produce secondary mRNA structure.

[0196] The protein of the present application can be changed in various ways, including amino acid substitution, deletion, truncation and insertion. The methods for such operations are generally known in the art. The methods for mutagenesis and polynucleotide changes are well known in the art. For guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein, it can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), which is incorporated herein by reference. Conservative substitutions such as replacing one amino acid with another amino acid having similar properties can be optimal.

[0197] It is expected that deletions, insertions, and substitutions of the protein sequences encompassed herein will not produce fundamental changes in the characteristics of the protein. However, where it is difficult to predict the exact effect of a substitution, deletion, or insertion, one skilled in the art will appreciate that the effect will be assessed by conventional screening assays. That is, activity can be assessed by the assays disclosed below.

[0198] Variant polynucleotides and proteins also include sequences and proteins derived from mutagenesis and recombination processes such as DNA shuffling. Strategies for such DNA shuffling are known in the art.

[0199] In this application, the term "amino acid" refers to any amino acid (both standard and non-standard amino acids), including but not limited to α-amino acids, β-amino acids, γ-amino acids and δ-amino acids. Examples of suitable amino acids include but are not limited to alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.

[0200] As used herein, the term "isolated" means removed from its natural environment or from other compounds present when the compound is first formed. The term "isolated" includes materials isolated from natural sources as well as materials (such as nucleic acids and proteins) recovered after production by recombinant expression in host cells, or chemically synthesized compounds such as nucleic acid molecules, proteins, and peptides. Any method of obtaining the proteins of interest is compatible with this application.

[0201] In this application, the term "expression cassette" refers to a DNA fragment that can be inserted into a nucleic acid or polynucleotide at a specific restriction site or by homologous recombination. As used herein, the DNA fragment comprises a polynucleotide encoding a polypeptide of interest, and the expression cassette and restriction sites are designed to ensure that the expression cassette is inserted into an appropriate reading frame for transcription and translation. In one embodiment, the expression cassette can include a polynucleotide encoding a polypeptide of interest and, in addition to the polynucleotide, also has elements that promote specific host cell transformation. In one embodiment, the expression cassette can also include elements that allow the expression of the polynucleotide encoding the polypeptide of interest to be enhanced in the host cell. These elements can include, but are not limited to, promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences, etc.

[0202] The expression cassette can also include a selective marker gene for selecting transformed cells. The selective marker gene is used to select the cells or tissues of transformation. The marker gene includes a gene encoding antibiotic resistance, such as the gene encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and a gene that confers resistance to herbicidal compounds (such as glufosinate, bromoxynil, imidazolinone and 2,4-dichlorophenoxyacetic acid ester (2,4-d)). Other selective markers include phenotypic markers such as beta-galactosidase and fluorescent proteins, such as green fluorescent protein (GFP), cyan fluorescent protein (CYP) and yellow fluorescent protein.

[0203] In this application, the terms "vector" and "construct", "cloning vector" and "expression vector" are used interchangeably, and mean a vector that can introduce a DNA or RNA sequence (e.g., an exogenous gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. "Non-viral vector" refers to any vector that does not contain a virus or retrovirus. In some embodiments, a "vector" is a DNA sequence that comprises at least one DNA replication origin and at least one selectable marker gene. Examples include, but are not limited to, plasmids, cosmids, bacteriophages, bacterial artificial chromosomes (BACs), or viruses that carry exogenous DNA into cells. Vectors can also include one or more genes, antisense molecules, and / or selectable marker genes and other genetic elements known in the art. Vectors can transduce, transform, or infect cells, thereby causing the cells to express nucleic acid molecules and / or proteins encoded by the vector. The term "plasmid" refers to a circular chain of nucleic acids that can replicate autosomes in prokaryotic or eukaryotic host cells. The term includes nucleic acids, which can be DNA or RNA, and can be single-stranded or double-stranded. The plasmid defined in this definition can also include sequences corresponding to bacterial replication origins.

[0204] In some embodiments, a "cloning vector" can replicate or integrate autonomously in the host cell genome and is further characterized by one or more restriction endonuclease sites at which the vector can be cut in a defined manner and a desired DNA sequence can be ligated into the vector so that the new recombinant plasmid retains its ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence can occur multiple times as the number of copies of the plasmid in the host cell (e.g., a bacterial host) increases, or only once per host before the host reproduces by mitosis. In the case of a bacteriophage, replication can occur actively during the lytic phase or passively during the lysogenic phase.

[0205] In some embodiments, an "expression vector" can be inserted into a desired DNA sequence by restriction enzyme digestion and ligation so that it is effectively linked to a regulatory sequence and can be expressed as an RNA transcript. The vector may also contain one or more marker sequences suitable for identifying whether a cell has been transformed or transfected by the vector. Markers include genes that encode proteins that increase or decrease their resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activity can be detected by standard methods known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes that have a visible effect on the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). Preferred vectors are those that are capable of autonomous replication and expression of the products of the structural genes present in the DNA fragments to which they are effectively linked.

[0206] In this application, the term "expression" refers to the biosynthesis of a gene product, including the transcription and / or translation of the gene product. "Expressing" or "producing" a protein or polypeptide from a DNA molecule refers to transcribing and translating the coding sequence to produce the protein or polypeptide, while "expressing" or "producing" a protein or polypeptide from an RNA molecule refers to translating the RNA coding sequence to produce the protein or polypeptide.

[0207] Gene expression can be influenced by external signals, such as exposure of a cell, tissue, or organism to a substance that increases or decreases gene expression. Gene expression can also be regulated anywhere along the pathway from DNA to RNA to protein. Regulation of gene expression can be achieved through control of transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after their production, or a combination thereof. The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally should include, as needed, 5′ non-transcribed and 5′ non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. In particular, such 5′ non-transcribed regulatory sequences will include a promoter region, which includes a promoter sequence that controls transcriptional control of an operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as needed. The vectors herein may optionally include a 5′ leader or signal sequence. The selection and design of appropriate vectors are within the ability and judgment of those skilled in the art.

[0208] In some embodiments, when the nucleic acid molecule encoding any enzyme of the present application is expressed in a cell, a variety of transcription control sequences (e.g., promoter / enhancer sequences) can be used to direct its expression. The promoter can be a natural promoter, i.e., the promoter of a gene in its endogenous environment, which provides normal regulation of gene expression. In some embodiments, the promoter can be constitutive, i.e., the promoter continuously transcribes its associated gene without regulation. A variety of conditional promoters can also be used, such as promoters controlled by the presence or absence of a molecule. Chemically regulated promoters can be used to regulate the expression of genes in a host by applying exogenous chemical regulators. Depending on the purpose, the promoter can be a chemically induced promoter, wherein the application of the chemical induces gene expression, or a chemically repressible promoter, wherein the application of the chemical represses gene expression. Chemically induced promoters are known in the art, including but not limited to the corn In2-2 promoter (which is activated by benzenesulfonamide herbicide safeners), the corn GST promoter (which is activated by hydrophobic electrophilic compounds used as pre-germination herbicides), and the tobacco PR-1a promoter (which is activated by salicylic acid). Other chemically regulated promoters of interest include glucocorticoid-inducible promoters among the steroid-responsive promoters, as well as tetracycline-inducible and tetracycline-repressible promoters.

[0209] Expression vectors containing all necessary expression elements are commercially available and 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, 1989. Cells are genetically engineered by introducing exogenous DNA (RNA) into the cells. The exogenous DNA (RNA) is placed under the effective control of transcriptional elements to allow expression of the exogenous DNA in the host cell.

[0210] As used herein, the term "transformation" includes all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to, transfection with viral vectors; transformation with plasmid vectors; electroporation; adipose infection; microinjection; Agrobacterium-mediated transfer; direct DNA uptake; WHISKERS TM mediated transformation; and microprojectile bombardment. These techniques can be used for both stable and transient transformation of host cells. "Stable transformation" refers to the introduction of a nucleic acid fragment into the genome of a host organism, resulting in genetic stability. Once stably transformed, the nucleic acid fragment is stably integrated into the genome of the host organism and any subsequent generations. Host organisms containing the transformed nucleic acid fragment are referred to as "transgenic" organisms. "Transient transformation" refers to the introduction of a nucleic acid fragment into the nucleus or DNA-containing organelles of a host organism, resulting in gene expression but not genetic stability.

[0211] In some embodiments, to transform hosts and host cells, the nucleotide sequences of the present application can be inserted into any vector known in the art that is suitable for expressing nucleotide sequences in hosts or host cells using standard techniques. The choice of vector depends on the preferred transformation technique and the target host species to be transformed. The transformation method depends on the host cell to be transformed, the stability of the vector used, the expression level of the gene product, and other parameters.

[0212] In the present application, term " plant " comprises seed, vegetable cell, plant protoplast, can be from the plant cell tissue culture, plant callus, plant piece of its regenerated plant and in plant or plant part such as embryo, pollen, ovule, seed, tuber, propagule, leaf, flower, branch, fruit, root, root tip, anther etc., be complete plant cell.The offspring, variant and mutant of regenerated plant are also included in the scope of the present application, and condition is that these parts comprise the polynucleotide of introducing.As used herein, unless otherwise clearly stated or apparent from the background of use, otherwise " offspring " and " offspring plant " comprise any subsequent generation of plant, no matter be produced by sexual reproduction and / or asexual reproduction.

[0213] The terms "transgenic plant" and "transformed plant" refer to equivalent terms for "plant" as described above, wherein the plant comprises a heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct introduced into the plant by, for example, any stable and transient transformation method disclosed elsewhere herein or otherwise known in the art. Such transgenic plants and transformed plants also refer, for example, to the plant into which the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct was first introduced, as well as any progeny plants thereof that comprise the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct.

[0214] In certain embodiments, a plant comprising a heterologous polynucleotide comprising an R gene nucleotide sequence of the present application is produced using the methods of the present application, the method involving genome editing to modify the nucleotide sequence of a natural or non-natural gene in the genome of the plant. Here, the natural or non-natural gene comprises a nucleotide sequence different from the R gene nucleotide sequence of the present application, and the modified natural or non-natural gene comprises the R gene nucleotide sequence of the present application. Generally, such methods include using a plant comprising a natural or non-natural gene in its genome, wherein the natural or non-natural gene comprises a nucleotide sequence homologous to the R gene nucleotide sequence of the present application, and further comprising introducing into the plant a nucleic acid molecule comprising at least a portion of the R gene nucleotide sequence of the present application. Preferably, the nucleotide sequence of the natural or non-natural gene has about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more nucleotide sequence identity with at least one R gene nucleotide sequence of the present application. Such a native or non-native gene may be, for example, a non-functional homologue of an R gene, in particular the Rpi-caj1 gene, which is not capable of conferring or is not known to confer resistance to plant diseases on plants.

[0215] The term "Solanaceae" is intended to include Solanaceae plants at any stage of maturity or development, and unless the context clearly indicates otherwise, any cell, tissue or organ (plant part) obtained or derived from any such plant. Solanaceae plant parts include, but are not limited to, fruits, stems, tubers, roots, flowers, ovules, stamens, leaves, embryos, meristematic regions, calli, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, etc. As used herein, the term "tuber" is intended to mean a whole tuber or any part thereof, such as a slice or part of a potato tuber comprising one or more buds (i.e., "eyes") suitable for field planting to produce potato plants. The application also includes seeds produced by the Solanaceae plants of the application.

[0216] In certain embodiments of the present application, the plants of the present application, particularly Solanaceae plants, may contain one, two, three, four, five, six or more nucleotide sequences encoding R proteins. Typically, but not necessarily, the two or more R proteins are different from one another. For the present application, when two R proteins have different amino acid sequences, the R protein is different from the other R protein. In certain embodiments of the present application, each of the different R proteins used to resist plant diseases caused by a certain species of Phytophthora has one or more differences in resistance characteristics, such as resistance to different species and / or groups of species of the same Phytophthora species or even different species of Phytophthora. By combining two, three, four, five, six or more nucleotide sequences with each nucleotide sequence encoding different R proteins for resistance to different species of a certain species of Phytophthora or certain species (spp.) of Phytophthora, a Solanaceae plant containing a broad spectrum of resistance to multiple species of a single species of Phytophthora or even multiple species of Phytophthora can be produced. Such Solanaceae plants, particularly potato or tomato plants, may be used in agriculture in areas where multiple races of a species of Phytophthora, such as multiple races of Phytophthora infestans, are prevalent.

[0217] Plants of the present application comprising multiple R genes can be produced, for example, by transforming a plant already comprising one or more other R gene nucleotide sequences with a heterologous polynucleotide comprising at least one Rpi-caj1 nucleotide sequence of the present application. Such plants already comprising one or more other R gene nucleotide sequences may comprise R genes native to the genome or plant, R genes introduced into the plant through sexual reproduction, or R genes introduced by transforming the plant or its ancestors with R gene nucleotide sequences. Alternatively, the one or more other R gene nucleotide sequences can be introduced into the plant of the present application, which already comprises the heterologous polynucleotide of the present application, by transformation or sexual reproduction, for example.

[0218] In other embodiments, two or more different R gene sequences can be introduced into a plant by stably transforming the plant with a heterologous polynucleotide or vector comprising two or more R gene nucleotide sequences. Alternatively, the heterologous polynucleotide of the present application can be integrated into the genome of the plant, adjacent to another R gene nucleotide sequence, using homologous recombination-based genome modification methods described elsewhere herein or known in the art.

[0219] In this application, the use of the term "DNA" or "RNA" is not intended to limit the application to polynucleotide molecules comprising DNA or RNA. It will be appreciated by those of ordinary skill in the art that the methods and compositions of the present application include polynucleotide molecules consisting of deoxyribonucleotides (i.e., DNA), ribonucleotides (i.e., RNA), or a combination of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs, including but not limited to nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, having binding properties similar to reference nucleic acids, and metabolized in a manner similar to reference nucleotides. Examples of such analogs include but are not limited to phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). The polynucleotide molecules of the present application also include all forms of polynucleotide molecules, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc. In addition, it will be understood by those of ordinary skill in the art that the nucleotide sequences disclosed herein also include the complementary sequences of the exemplary nucleotide sequences.

[0220] Table 1 Species information of the genes in this application

[0221] Table 2 Sequence information of genes and proteins in this application

[0222] Unless otherwise specified, all reagents, instruments, strains, and biological materials used in this application can be obtained through commercial channels. The Phytophthora infestans strains JH19, NL07434, 88069, T30-4, EC3527, HB1501, HN1602, FJ20021, SD21143, LB21023, AH1624, CQ1906, HN1403, YN1407, AH1618, YN1414, and MG1924 used in the following examples were obtained from the Crop Disease Genomics Laboratory, Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University.

[0223] The present application is further described below with reference to the embodiments:

[0224] Example 1

[0225] 1. Cloning of the Rpi-caj1 gene

[0226] Based on the nucleotide sequence of the Rpi-caj1 gene, primers were designed to amplify the Rpi-caj1 gene. Using gDNA from wild potato leaves as a template, the Rpi-caj1 gene was amplified by PCR. The PCR product was recovered by gel excision and ligated into the linearized pBin307 vector digested with SmaI. The recombinant plasmid was cloned and sequenced using the ClonExpress Ultra One Step Cloning Kit. The positive recombinant plasmid was named pBin307-Rpi-caj1.

[0227] Primer sequences are shown in the table below.

[0228] Table 3 Primer sequences Note: * indicates that the corresponding primer pair is used for cloning genes containing promoters.

[0229] PCR reaction system and conditions:

[0230] PCR reaction system: 0.2 μM, 1× PCR buffer, 0.2 mM dNTPs, 3.75 mM MgCl2, and 2 U of DNA polymerase (TaKaRa Biotechnology, Dalian) per 25 μL reaction system.

[0231] PCR reaction conditions: step 1, 94°C, 5 min; step 2, 94°C, 1 min; step 3, 56°C, 2 min; step 4, 72°C, 2 min; step 2 to step 4 are cycled 35 times; step 6, 72°C, 10 min; step 7, 4°C, constant temperature.

[0232] Enzyme digestion system and conditions:

[0233] Enzyme digestion reaction system: restriction endonuclease, 2 units; plasmid DNA, 1 μg; 10X NEBuffer, 1 μL (1X); total reaction system: 20 μL.

[0234] Enzyme digestion reaction conditions: The digestion temperature of SmaI enzyme was 25°C and the incubation time was 60 minutes.

[0235] Recombination system and conditions:

[0236] Recombination reaction system: linearized plasmid, 1 μL; target gene PCR product, 2 μL; 2× ClonExpress Mix, 5 μL; total reaction system: 10 μL.

[0237] Recombination reaction conditions: temperature: 50°C, time: 5 minutes.

[0238] 2. Transform the plasmid homologous recombination product into E. coli using the following method:

[0239] 1). Adjust the temperature of the constant temperature water bath to 42℃ in advance.

[0240] 2) Take out a tube (100 μL) of competent DH5α E. coli from a -70°C freezer, immediately melt it with your fingers, and then place it on ice. Keep it in an ice bath for 5-10 minutes.

[0241] 3). Add 5 μL of recombinant plasmid mixture (DNA content not exceeding 100 ng), shake gently and place on ice for 20 minutes.

[0242] 4). After gently shaking, place the tube in a 42°C water bath for 1-2 minutes for heat shock, then quickly return it to ice and let it stand for 3-5 minutes.

[0243] 5) In a clean bench, add 500 μL of LB medium (without antibiotics) to each of the above tubes, mix gently, and then fix them to the spring frame of a shaker and shake at 37°C for 1 hour.

[0244] 6) In a clean bench, take 100-300 μL of the above transformation mixture and drop it into a solid LB plate culture dish containing 50 μg / mL kanamycin, and spread it evenly with a glass coating rod burned by an alcohol lamp.

[0245] 3. Use the FastPure Plasmid Mini Kit to extract the plasmid.

[0246] 4. Competent plasmid transformation of Agrobacterium:

[0247] 1) Add 5 to 10 μL of prepared plasmid DNA to 100 μL of Agrobacterium GV3101 competent cells.

[0248] 2) Incubate the Agrobacterium-plasmid mixture in a 37°C water bath for 5 minutes.

[0249] 3) Add 1 ml of LB liquid medium to the Agrobacterium-plasmid mixture and culture at 28°C with shaking for 2-4 hours.

[0250] 4). Centrifuge the Agrobacterium (10000 g, 30 seconds), remove 0.9 mL of the supernatant, and resuspend the Agrobacterium cells in the remaining supernatant.

[0251] 5) Spread the suspension evenly on a plate containing 50 μg / mL kanamycin or LB solid medium. Incubate at 28°C for 2 days.

[0252] Example 2 Transient Expression of Rpi-caj1-C534 in Nicotiana benthamiana Leaves and Inoculation with Phytophthora infestans

[0253] 100 μL of the Agrobacterium culture prepared in Example 1 was taken from -80°C and placed in a 1.5 mL centrifuge tube. 1 mL of LB (kana + Rif) liquid medium was added. The culture was shaken at 28°C, 220 r. for 12 h. Then 100 μL of the culture was taken in a 50 mL centrifuge tube. 10 mL of LB (kana + Rif) liquid medium was added. The culture was shaken at 28°C, 220 r. for overnight. The OD value was measured. 600 About 0.5.

[0254] The overnight culture of Agrobacterium containing the Rpi-caj1 binary expression vector pBin307-Rpi-caj1 was collected by centrifugation, resuspended in MES buffer, and the OD 600 = 0.3, injected into one side (left side) of a 4-week-old wild-type Nicotiana benthamiana leaf. Agrobacterium containing pBin307-gfp was injected into the other side (right side) of the leaf as a negative control. One day after injection, 10 μL droplets of 200,000 to 400,000 spores / mL of Phytophthora infestans JH19 zoospores were inoculated onto both sides of the leaf. Results were counted 5 days after inoculation (dpi). Resistance was assessed by direct observation of lesions on the leaves; resistance was observed by the absence of lesions.

[0255] The results showed that under the CaMV35S promoter, Rpi-caj1 was able to produce weak, punctate necrosis in N. benthamiana leaves, but the large-scale necrosis caused by infection with P. infestans zoospores was significantly reduced on the side where Rpi-caj1 was transiently expressed. This indicates that transient expression of Rpi-caj1 in N. benthamiana leaves can confer significant resistance to P. infestans zoospore inoculation (Figure 1).

[0256] Example 3 Transient expression of Rpi-caj1 homologous genes from different sources in tobacco leaves and inoculation with Phytophthora infestans

[0257] The method is the same as Example 2.

[0258] Compared to the GFP control, leaves transiently expressing Rpi-caj1-C534, Rpi-caj1-C813, Rpi-caj1-C550, Rpi-caj1-C509, Rpi-caj1-C450, and Rpi-caj1-C419 showed significant resistance to Phytophthora infestans. These results demonstrate that the Rpi-caj1 homologs from these diverse sources can confer resistance to Phytophthora infestans (Figure 2).

[0259] Example 4 Transformation of Rpi-caj1-C534 into the Potato Genome

[0260] In this example, the disease resistance gene Rpi-caj1 is driven by the Pcaj promoter, a disease resistance gene derived from wild potato, and the nptII gene is driven by the CamV 35S promoter. These genes are constructed into the plant expression vector pBin307 (same as in Example 1). Agrobacterium-mediated stable transformation of the stems of the susceptible potato variety Désirée was used to obtain transgenic plants. Kanamycin resistance screening and phenotypic verification revealed transgenic material resistant to late blight and resistant to the herbicide kanamycin. The specific transgenic method is as follows:

[0261] (1) Stem segment pre-culture:

[0262] Z1N2 medium was covered with two sterilized filter papers, and 2 mL of PACM (plant MS liquid medium containing 1 μg / mL 2,4-dichlorophenoxyacetic acid and 0.5 μg / mL kinetin) was added. A certain number of stem segments were cut, arranged neatly, and cultured under light (48 h).

[0263] (2) Bacteria activation:

[0264] Take 100 μL of Agrobacterium culture liquid from -80℃, put it in a 1.5mL centrifuge tube, add 1mL LB (kana + Rif) liquid medium, and culture it on a shaker at 28℃, 220r for 12h. Then take 100 μL of it in a 50mL centrifuge tube, add 10mL LB (kana + Rif) liquid medium, and culture it on a shaker at 28℃, 220r overnight. Detect the OD value. The OD value is required. 600 About 0.5.

[0265] (3) Infection:

[0266] The Agrobacterium culture liquid was centrifuged (4°C, 4000r, 10min), the bacterial precipitate was collected, and the OD was resuspended in MS20 liquid (1‰ acetosyringone AS was added, and 10μL AS was added to each 10mL liquid MS20 medium). 600 To 0.5. Place the pre-cultured stem segments in the resuspended bacterial solution for 10 minutes, shaking continuously. Take the stem segments and culture them in Z1N2 AS medium (covered with filter paper) at 24°C in the dark for 48 hours.

[0267] (4) Differentiation:

[0268] Stem segments were cultured in differentiation medium Z2N0.01. The medium was changed every two weeks. When calli differentiated and sprouted, they were transferred to rooting medium (MS30 + 2 mL 300 mg / mL Timentin TMT + 1 mL 50 mg / mL kanamycin per liter) and subcultured every 3-4 weeks.

[0269] (5) Screening of transgenic positive plants

[0270] The potato seedlings on the rooting medium can be screened after they have taken root and grown to a certain size. Leaves of the plants are taken and DNA is extracted for PCR verification.

[0271] Primers were designed based on the Rpi-caj1 gene sequence. PCR amplification was performed using genomic DNA from transgenic potato leaves as a template. Reaction conditions were: 95°C for 5 minutes, 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 2 minutes, Go to 2 for 30 cycles, and 72°C for 8 minutes. The PCR product was approximately 350 base pairs. The results confirmed that the target gene, Rpi-caj1, was integrated into the potato genome (Figure 3).

[0272] Example 5 Inoculation of transgenic potato leaves with Phytophthora infestans

[0273] In this example, late blight resistance in transgenic potatoes was assessed using zoospores of the P. infestans strain collected from culture dishes. 10 μL of P. infestans zoospores, containing 200,000 to 400,000 spores per mL, were inoculated onto detached leaves from 4- to 6-week-old potato plants obtained using the method of Example 4. Results were recorded five days after inoculation. Eight separate leaves were used for each inoculation experiment, and each experiment was replicated at least three times.

[0274] The results showed that compared with untransformed potatoes (Désirée), transgenic potato plants stably expressing Rpi-caj1-C534 showed significant resistance to 16 different infestative Phytophthora strains (NL07434, 88069, T30-4, EC3527, HB1501, HN1602, FJ20021, SD21143, LB21023, AH1624, CQ1906, HN1403, YN1407, AH1618, YN1414, MG1924). Its resistance spectrum was superior to that of the known disease resistance gene Rpi-blb2, demonstrating that it can mediate broad-spectrum resistance of potato to late blight (Figure 4).

[0275] The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A nucleic acid molecule, characterized in that It contains at least one of the following nucleotide sequences: (a1) the nucleotide sequence shown in SEQ ID NO: 1; (a2) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:7; (a3) the nucleotide sequence shown in SEQ ID NO: 13; (a4) a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in any one of (a1) and (a3); (a5) A nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with any of the amino acid sequences shown in (a2).

2. The nucleic acid molecule according to claim 1, characterized in that In (a4), the nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 includes at least one of the nucleotide sequences shown in SEQ ID NO: 2 to 6; and / or, in (a4), the nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 13 comprises at least one of the nucleotide sequences shown in SEQ ID NO: 14 to 18; And / or, in (a5), the nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 includes at least one of the nucleotide sequences shown in SEQ ID NOs: 8 to 12.

3. A nucleic acid molecule composition, characterized in that: It comprises the nucleic acid molecule according to claim 1 or 2 and another R gene; Optionally, the additional R genes include at least one of the following genes: Rpi-amr3i, Rpi-blb1, Rpi-sto1, Rpi-pta1, Rpi-blb2, Rpi-blb3, Rpi-abpt, R2-like, R2, Rpi-edn1.1, Rpi-edn1.2, Rpi-snk1.1, Rpi-snk1.2, Rpi-hjt1.1-Rpi-hjt1.3, Rpi-bt1, R1, R3a, R3b, Rpi-vnt1.1, Rpi-vnt1.2, Rpi-vnt1.3, Rpi-mcq1, Rpi-chc, Ph-3 or R8.

4. A biomaterial, characterized in that: The biological material is any one of the following (b1) to (b3): (b1) an expression cassette or an expression cassette composition comprising the nucleic acid molecule according to claim 1 or 2 or the nucleic acid molecule composition according to claim 3; (b2) a vector or vector composition containing the nucleic acid molecule according to claim 1 or 2 or the nucleic acid molecule composition according to claim 3, or a vector or vector composition containing the expression cassette or expression cassette composition according to (b1); (b3) a host cell containing the nucleic acid molecule of claim 1 or 2 or the nucleic acid molecule composition of claim 3, or a host cell containing the expression cassette or expression cassette composition of (b1), or a host cell containing the vector or vector composition of (b2); optionally, the host cell comprises a bacterial or fungal cell.

5. The biomaterial according to claim 4, characterized in that The expression cassette or expression cassette composition further comprises an operably linked promoter; Optionally, the promoter includes an operably linked endogenous promoter and / or an operably linked heterologous promoter.

6. A promoter, characterized in that It comprises at least one of the following nucleotide sequences: the nucleotide sequence shown in SEQ ID NO: 19, and / or a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence shown in SEQ ID NO:

19.

7. The promoter according to claim 6, characterized in that The nucleotide sequence having at least 75% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 includes at least one of the nucleotide sequences shown in SEQ ID NOs: 20-24.

8. A method for increasing the resistance of plants to plant diseases caused by at least one subspecies of at least one species of the genus Phytophthora, characterized in that The method comprises modifying at least one plant cell to contain a heterologous polynucleotide comprising the nucleic acid molecule of claim 1 or 2 or the nucleic acid molecule composition of claim 3.

9. The method according to claim 8, characterized in that The method further comprises regenerating the plant cell into a plant comprising the heterologous polynucleotide in its genome.

10. A method for controlling plant diseases caused by at least one subspecies of at least one species of Phytophthora in crop production, characterized in that: The method comprises: Modifying at least one plant cell to contain a heterologous polynucleotide comprising the nucleic acid molecule of claim 1 or 2 or the nucleic acid molecule composition of claim 3; regenerating the plant cell into a plant comprising the heterologous polynucleotide in its genome; The seedlings, tubers or seeds of the plant are planted and cultivated under conditions conducive to the growth and development of the plant.

11. A method for detecting plant disease resistance, characterized in that: The method comprises detecting the presence of the nucleic acid molecule of claim 1 or 2 or the nucleic acid molecule composition of claim 3 in a plant, a plant part thereof or a plant cell.

12. The method according to claim 11, characterized in that The method for detecting the presence of the nucleic acid molecule described in claim 1 or 2 or the nucleic acid molecule composition described in claim 3 comprises: detecting the presence of the nucleic acid molecule or the nucleic acid molecule composition by detecting the complete nucleic acid molecule or the nucleic acid molecule composition, or detecting at least one molecular marker in the nucleic acid molecule or the nucleic acid molecule composition.

13. The method according to claim 11 or 12, characterized in that: The method for detecting the presence of the nucleic acid molecule of claim 1 or 2 or the nucleic acid molecule composition of claim 3 comprises PCR amplification, nucleic acid sequencing, nucleic acid hybridization or an immunological assay for detecting a protein or polypeptide encoded by the nucleic acid molecule or nucleic acid molecule composition.

14. The method according to any one of claims 8 to 13, characterized in that: The plants include plants of the Solanaceae family; Preferably, the Solanaceae plant includes but is not limited to at least one of potato, tomato, eggplant, pepper, tobacco, petunia, tomatillo, and cape gooseberry; Preferably, the Solanaceae plant is potato and / or tomato.

15. A method for selecting plants, characterized in that The method comprises: detecting the presence of the nucleic acid molecule of claim 1 or 2 or the nucleic acid molecule composition of claim 3 in a plant, a plant part thereof or a plant cell; selecting a plant comprising at least one copy of the nucleic acid molecule or the nucleic acid molecule composition in its genome; Preferably, the method comprises using primers shown in SEQ ID NOs: 25-38.

16. A protein, characterized in that It contains at least one of the following amino acid sequences: (e1) the amino acid sequence shown in SEQ ID NO:7; (e2) an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1; (e3) an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 13 or a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 13; (e4) an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, wherein a protein comprising the amino acid sequence is capable of conferring resistance to plant diseases caused by at least one subspecies of at least one species of the genus Phytophthora to a plant comprising the protein; (e5) a fusion amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 7; (e6) The amino acid sequence shown in SEQ ID NO: 7 has one or more amino acid residues substituted and / or deleted and / or added The amino acid sequence with the same function is obtained.

17. The protein according to claim 16, characterized in that (e2), the nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 includes at least one of the nucleotide sequences shown in SEQ ID NO: 2 to 6; and / or, the nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 13 comprises at least one of the nucleotide sequences shown in SEQ ID NO: 14 to 18; And / or, the amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 comprises at least one of the nucleotide sequences shown in SEQ ID NOs: 8-12.

18. A plant or a plant part thereof, characterized in that The plant includes a transgenic plant containing the nucleic acid molecule according to claim 1 or 2 or the nucleic acid molecule composition according to claim 3.

19. A method for producing a plant, characterized in that The method comprises transforming a plant with at least one of the nucleic acid molecule of claim 1 or 2 or the nucleic acid molecule composition of claim 3, the expression cassette or expression cassette composition of claim 4, the vector or the vector composition.

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