Application of COP1 gene mutation in plant disease resistance

Abstract

The present invention relates to the field of plant molecular biology, and in particular to the use of plant COP1 gene mutants for combating plant diseases. Substitution of one amino acid among 16 amino acids that are sterically close within a range of 5 Å centered on S648 in the Arabidopsis COP1 protein affects the accumulation of the EDS1 protein and specifically enhances the resistance of plants to diseases such as powdery mildew through the SA pathway.

Description

Technical Field

[0001] The present invention relates to the field of plant molecular biology. Specifically, the present invention relates to the use of plant COP1 gene mutants in plant disease resistance. Background Art

[0002] In nature, plants are exposed to a variety of pathogens. Most plants cannot effectively avoid pathogen infection by moving their spatial positions. Therefore, plants have evolved complex defense mechanisms to sense pathogens and initiate immune defenses. The immune defense of plants against pathogens is usually that pathogen / microbe-associated molecular patterns (PAMPs / MAMPs) are recognized by pattern recognition receptors (PRRs) located on the plant cell plasma membrane, that is, pattern-triggered immunity (PTI). In addition, in order to further infect plants, pathogens will produce virulence proteins (effectors) that inhibit plant PTI, interfering with the effective recognition of PAMPs / MAMPs by plant PRRs. And plants will further evolve resistance proteins (R proteins) that can specifically recognize pathogen effectors, and control the spread of pathogens by causing cell death through a hypersensitive response (HR) at the site of pathogen infection, that is, effector-triggered immunity (ETI).

[0003] Plant hormones are also involved in the defense against pathogens. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) have been widely reported in plant-pathogen interactions. The ability of plants to resist pathogen invasion depends on the crosstalk between different hormone signaling pathways. SA / JA signaling molecules show different response strategies in plant defense depending on the type of invading pathogen, but the crosstalk between the two is involved in the plant defense response. SA is a key hormone for resisting biotrophic and hemibiotrophic pathogens. JA-dependent defense is usually triggered by necrotrophic pathogens. These two signaling pathways affect each other through a complex network of synergistic and antagonistic effects.

[0004] SA is a small phenolic compound widely present in plants, regulating various physiological processes and adaptive stress responses. Thirty years ago, based on its impact on pathogen infection, SA was reported to be involved in plant immune responses and has since received extensive attention in the field of plant-pathogen interactions. Many studies on SA-mediated plant defense have shown that induced SA accumulates not only at the infected site but also in uninfected tissues. That is, SA is not only a necessary condition for inducing the hypersensitive response (HR) but also crucial for establishing local or systemic resistance. SA induces an increase in the expression of defense-related (PR) genes and leads to the burst of reactive oxygen species (ROS) and the deposition of callose during pathogen infection.

[0005] Through forward genetic screening of Arabidopsis mutants that cannot activate the expression of PR genes, the antibacterial protein NPR1 (Nonexpresser of pathogenesis-related (PR) genes 1) was discovered. Similar to NPR3 and NPR4, NPR1 binds to SA and serves as a receptor for SA. Before pathogen infection, NPR1 exists as an oligomer in the cytoplasm, which is crucial for maintaining protein homeostasis. When pathogens infect, the redox reaction induced by SA reduces the oligomeric NPR1 to an active monomer, and the NPR1 monomer enters the nucleus. As a transcriptional coactivator, NPR1 interacts with TGA (TGACG-binding motif) and TCP (teosinte branched 1 / cycloidea / PCF) transcription factors and activates the expression of PR genes. In addition to PR genes, NPR1 also controls the expression of most SA-responsive genes.

[0006] The EDS1 family includes EDS1, PAD4, and SAG101. They are a class of lipase-like proteins localized in the nucleus and cytoplasm, which can promote host cell death within the ETI immune pathway and transcriptionally activate the immune defense pathway during basal immune responses. EDS1 physically interacts with PAD4, SAG101, and some other R proteins, and different EDS1-PAD4 and EDS1-SAG101 protein complexes are essential for different R protein-mediated ETI. In addition, EDS1 and NPR1 interact to form a protein complex and synergistically activate plant immunity through the SA signaling pathway. After SA induction, NPR1 directly recruits EDS1 to the PR1 promoter to promote the expression of the PR1 gene. And a positive feedback loop is formed between the two, that is, NPR1 directly upregulates the transcription of EDS1, and EDS1 stabilizes NPR1 in plant-pathogen interactions.

[0007] Light is not only an energy source for photosynthetic plants, but also a signaling molecule that regulates plant growth and development. At least five different photoreceptors are responsible for perceiving light signals of different wavelengths. After being activated by light, photoreceptors initiate complex and delicate molecular networks that mediate most light-dependent developmental processes in plants, including skotomorphogenesis (etiolation) and photomorphogenesis (de-etiolation). Previous research results have shown that recessive constitutively photomotphogenic / de-etiolated / fusca (cop / det / fus) mutants exhibit constitutive photomorphogenic phenotypes even in the dark. COP / DET / FUS forms different protein complexes with other components, namely the COP1-SPA complex, the CDD complex, and the COP9 signalosome, all of which promote skotomorphogenesis and inhibit photomorphogenesis in the dark.

[0008] COP1 is a core repressor of photomorphogenesis. Null mutations in COP1 are lethal at the seedling stage, while weak mutants cop1-4 and cop1-6 also exhibit constitutive photomorphogenic developmental phenotypes in the absence of light signals. As an E3 ubiquitin ligase, COP1 mediates the degradation of downstream target proteins through the 26S proteasome system. COP1 accumulates in the nucleus under dark conditions and ubiquitinates and degrades some substrates. Under light, photoactivated phyA, phyB, CRY1, and CRY2 inhibit the activity of the COP1-SPA complex. As a long-term strategy, under long-term light conditions, COP1 is redistributed from the nucleus to the cytoplasm, and thus its activity is also inhibited in the long term.

[0009] Photoperiod strongly affects the phenology and physiological responses of plants and plays an important role in regulating plant defense responses, such as regulating the lesion mimic mutant phenotypes of plants and resistance to pathogens. The key light signaling components De-Etiolated1 (DET1) and Constitutive Photomorphogenic 1 (COP1) negatively regulate immunity, are crucial for the immune regulation of photoperiod and temperature, and are regulated by the transcription factor Phytochrome Interacting Factor 4 (PIF4).

[0010] Powdery mildew (English name: Mulberry powdery mildew), also known as white back disease and mulberry powdery mildew, is a disease caused by fungi in the family Erysiphaceae and is a common crop plant disease. This disease is exogenous, grows on the leaf surface, forms gray spots, absorbs plant water and nutrients, seriously affects photosynthesis, hinders leaf growth, and can cause the plant to completely stop growing, resulting in premature senescence and loss of yield in severe cases. Therefore, finding plant varieties resistant to powdery mildew is an urgent problem in this field.

[0011] Under dark conditions, COP1 interacts with EDS1, ubiquitinates and degrades EDS1. The inventors of the present invention found that after the serine at position 648 of COP1 was replaced with asparagine (referred to as cop1-21), the accumulation level of EDS1 was higher than that of the wild-type Col-0. And in the inoculation experiment, compared with the wild-type Col-0, this mutant can specifically enhance the resistance of plants to powdery mildew without affecting normal development. Therefore, the COP1 gene variant of the present invention has broad application value, and the COP1 genes of many plant species can be mutated similar to the present invention to cultivate many plant varieties with disease-resistant characteristics. Summary of the Invention

[0012] The inventors of the present invention found that a region within 5 Å centered on S648 in the three-dimensional structure of the Arabidopsis COP1 protein (hereinafter referred to as the 5 Å CO-binding region) is a key region that can confer resistance to powdery mildew in Arabidopsis. Through research, it was found that the change in the structure of the 5 Å CO-binding region caused the accumulation of the EDS1 protein, thereby improving the resistance of plants to EDS1-related diseases, and the change in the structure of the 5 Å CO-binding region had no effect on the growth and development of plants. The mechanism for the above effects is that after amino acid substitution occurs in the amino acids within the 5 Å CO-binding region, only very small local changes in the 5 Å CO-binding region are affected, such as Figure 3 and Figure 4 shown, while the structures of other parts are not affected. Therefore, an amino acid substitution in the 5 Å CO-binding region usually produces the above effects of resistance to EDS1-related diseases without affecting growth and development, especially a substitution of the same type of amino acid in the 5 Å CO-binding region.

[0013] Accordingly, one aspect of the present invention relates to the use of a COP1 variant protein or its encoding nucleic acid molecule in plant EDS1-related diseases, wherein the COP1 variant protein is a variant of the COP1 protein of angiosperm plants as follows: one amino acid at positions I373, V374, S375, S376, Y554, F595, V596, G597, F646, I647, S648, A649, V650, A662, N663, S664 in the amino acid sequence of SEQ ID NO: 2 is replaced, or based on the protein sequence alignment with SEQ ID NO: 2, one amino acid corresponding to positions I373, V374, S375, S376, Y554, F595, V596, G597, F646, I647, S648, A649, V650, A662, N663, S664 in other angiosperm COP1 proteins is replaced; the nucleic acid molecule can be any form of nucleic acid molecule encoding the above COP1 variant protein, such as DNA, cDNA or mRNA.

[0014] In a more preferred embodiment, the amino acid substitution in the 5 Å CO-binding region is a conservative amino acid substitution.

[0015] More preferably, the conservative amino acid substitution is that serine at position S648 of SEQ ID NO: 2, or based on the protein sequence alignment with SEQ ID NO: 2, serine at the position corresponding to S648 of SEQ ID NO: 2 in other angiosperm COP1 proteins is replaced by a conservative amino acid, such as serine is replaced by asparagine, glutamine, tyrosine, threonine, cysteine or glycine.

[0016] Most preferably, the conservative substitution is serine replaced by asparagine.

[0017] In the most preferred embodiment, the COP1 variant protein has the sequence shown in SEQ ID NO.4, and the nucleic acid molecule has the sequence shown in SEQ ID NO.3.

[0018] The amino acid sequence of the 5 Å CO-binding region of the COP1 protein is highly conserved in many plants studied, such as plants in the families Cruciferae, Rosaceae, Leguminosae, Gramineae, Solanaceae, Umbelliferae, Compositae, Malvaceae, or Cucurbitaceae, with an identity of 100%. In addition, in plants of the family Cruciferae such as Arabidopsis, Arabis, Eutrema, Camelina, Capsella, Raphanus, Sinapis, or Brassica, in addition to the 100% identity of the amino acid sequence of the 5 Å CO-binding region in the COP1 protein, the amino acid sequence of the entire WD40 domain is also highly conserved. Therefore, in the technical solution of the present invention, the angiosperm plants are plants of the family Cruciferae, Rosaceae, Leguminosae, Malvaceae, Cucurbitaceae, Compositae, Solanaceae, Umbelliferae, or Gramineae. Further, the Cruciferous plants are Arabidopsis thaliana of the genus Arabidopsis, Arabis alpina of the genus Arabis, Eutrema salsugineum of the genus Eutrema, Camelina sativa of the genus Camelina, Capsella rubella of the genus Capsella, Raphanus sativus of the genus Raphanus, Sinapis alba of the genus Sinapis, Brassica oleracea of the genus Brassica, Brassica cretica, Brassica rapa, or Brassica napus.Furthermore, the Rosaceae plants are strawberry (Fragaria ananassa), Chinese rose (Rosa chinensis), apple (Malus domestica) or peach (Prunus persica); the Leguminosae plants are soybean (Glycine max) or peanut (Arachis hypogaea); the Gramineae plants are wheat (Triticum aestivum), barley (Hordeum vulgare), corn (Zea mays), sorghum (Sorghum bicolor), japonica rice (Oryza sativa Japonica Group) or indica rice (Onyza sativa Indica Group); the Solanaceae plants are tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum) or potato (Solanum tuberosum); the Umbelliferae plant is carrot (Daucus carota subsp. Sativus); the Compositae plant is sunflower (Helianthus annuus); the Malvaceae plant is cotton (Gossypium hirsutum); and the Cucurbitaceae plant is cucumber (Cucumis sativus).

[0019] In another aspect of the present invention, the plant disease is a plant EDS1-related disease. Preferably, the plant EDS1-related disease is powdery mildew caused by the infection of fungi Erysiphales, downy mildew caused by the infection of fungi Peronosporales, Phytophthora blight caused by the infection of fungi Phytophthora, Alternaria leaf spot caused by the infection of fungi Alternaria, bacterial wilt caused by the infection of bacteria Ralstonia solanacearum, tomato bacterial leaf spot caused by Pseudomonas syringae, tobacco wildfire disease, snapdragon blight, bacterial leaf spot, wheat bacterial glume blotch, berberis bacterial leaf spot, cannabis bacterial blight, bacterial blight, halo blight, delphinium black spot, bacterial leaf spot, soybean bacterial blight (phytophthora), cherry and plum canker, bacterial angular leaf spot of cucurbits, coffee halo blight, bacterial black spot, bayberry cancer, millet brown stripe, apple blister, peach bacterial blight, bean halo blight, pea bacterial blight, bacterial leaf spot, oat stripe disease, syringa blight, marigold bacterial leaf spot, camellia branch blight, kiwifruit canker, head rot or platycodon leaf spot.

[0020] Those skilled in the art can easily make appropriate adjustments to the technical solutions of the present invention according to specific needs. These adjustments can still solve the technical problems of the present invention and are also within the scope of protection of the claims of the present invention.

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the gene structure of Arabidopsis thaliana COP1 gene. Among them, the gene structure is drawn according to the Arabidopsis thaliana COP1 gene sequence AT2G32950. The figure shows the gene structure of Arabidopsis thaliana COP1, including 5'-UTR, exons, introns and 3'-UTR, as well as the position of the nucleotide encoding the S648N mutation site, that is, the 1943rd nucleotide in the CDS coding region is mutated from G to A.

[0023] Figure 2 It is a schematic diagram of the protein structure of Arabidopsis thaliana COP1. It shows three domains of the COP1 protein, which are the RING finger domain, the coiled coil domain and seven WD40 repeat sequences from the N-terminus to the C-terminus. At the same time, it also shows the mutation sites involved in the cop1-4 and cop1-6 mutants in the art, as well as the S648N (named cop1-21 in the present invention) mutation site of an embodiment of the present invention.

[0024] Figure 3 It is a schematic diagram of the hydrogen bonds formed by S648 in the three-dimensional structure of Arabidopsis thaliana COP1 protein. Among them, the side chain -OH in S648 forms two hydrogen bonds with -C=O of V374 and -C=O of A662, and the -NH2 of S648 forms a hydrogen bond with -C=O of A662.

[0025] Figure 4 It is a schematic diagram of the hydrogen bonds formed by N648 in the three-dimensional structure of the variant protein of Arabidopsis thaliana COP1. The side chain in N648 does not form hydrogen bonds with the surrounding amino acids, and only the -NH2 of N648 forms a hydrogen bond with -C=O of A662.

[0026] Figure 5 It is a schematic diagram of the amino acids within 5 Å centered on S648 in Arabidopsis thaliana COP1 and the hydrogen bonds formed. The amino acids within 5 Å centered on S648 are I373, V374, S375, S376, Y554, F595, V596, G597, F646, I647, A649, V650, A662, N663, S664, and 12 hydrogen bonds are formed in this region.

[0027] Figure 6Show the alignment results of COP1 protein sequences from different plants. Among them, the WD40 region of COP1 in each plant is relatively conserved compared to other regions, and the amino acids in the 5 Å CO-binding region are exactly the same. Each lamellar region of the 7 blades is marked with an underline in the figure, and the amino acids in the 5 Å CO-binding region of the present invention are marked in bold.

[0028] Figure 7 It is a figure showing the alignment results of COP1 protein sequences of cruciferous plants. Among them, the amino acid sequence alignment results of the WD40 domain (amino acids 349 to 675 in Arabidopsis thaliana as an example) show 95.7% identity, and the 16 amino acids in the 5 Å CO-binding region are completely conserved, with 100% identity. The amino acids in the 5 Å CO-binding region of the present invention are marked in bold.

[0029] Figure 8 It is a figure comparing the plant phenotypes of wild-type Col-0 and mutants cop1-21, cop1-4, cop1-6 of Arabidopsis thaliana. Among them, A is a photo of the plant type, and B is a violin plot of the fresh weight calculated based on the plant type in A. It can be seen that cop1-21 has no obvious adverse effect on the growth and development of plants, and even compared with Col-0, its fresh weight has a significant increase.

[0030] Figure 9 It is a photo of the leaf infection situation after quantitative inoculation of powdery mildew on wild-type Col-0 and mutants cop1-21, cop1-4, cop1-6, overexpression material 35S::COP1-GFP of Arabidopsis thaliana, showing that there is a large amount of white powder on the surface of Col-0 leaves, no visible powder on the surfaces of cop1-21, cop1-4, cop1-6 leaves, and a large amount of white powder on the surface of 35S::COP1-GFP leaves.

[0031] Figure 10 It is a photo of trypan blue staining of leaves after quantitative inoculation of powdery mildew on wild-type Col-0 and mutants cop1-21, cop1-4, cop1-6, and 35S::COP1-GFP of Arabidopsis thaliana. Compared with Col-0, Arabidopsis thaliana cop1-21, cop1-4, cop1-6 have fewer conidiophores, and 35S::COP1-GFP Arabidopsis thaliana has more conidiophores.

[0032] Figure 11 It is based on Figure 10 Quantification diagram of conidiophores calculated from trypan blue staining, where different lowercase letters on the bar graph represent significant differences. It can be seen that compared with Col-0, Arabidopsis thaliana cop1-21, cop1-4, cop1-6 have significantly fewer conidiophores, and 35S::COP1-GFP Arabidopsis thaliana has significantly more conidiophores.

[0033] Figure 12It is COP1 S648N Results of the co-immunoprecipitation experiment with EDS1. Among them, the EDS1 protein was detected by hybridization with an anti-HA antibody, and the COP1 and variant proteins, as well as the individual GFP protein, were detected by hybridization with an anti-GFP antibody. The results showed that a large amount of the EDS1 protein could co-precipitate with the COP1 protein.

[0034] Figure 13 It is a Western blot of the EDS1 protein abundance in wild-type Arabidopsis Col-0 and mutant cop1-21 and eds1-2 plants before and after infection with the fungal powdery mildew G. cichoracearum. Among them, the EDS1 protein was detected by hybridization with an anti-EDS1 antibody, and the ACTIN protein was detected by hybridization with an anti-ACTIN antibody to show the consistency of the loading amount during electrophoresis. The numbers under the EDS1 band are the amounts of the EDS1 protein calculated based on the band density, with the amount of the EDS1 protein in the wild-type Col-0 plants taken as 1.

[0035] Figure 14 It is the content levels of the plant hormones jasmonic acid (JA), jasmonoyl-isoleucine (JA-Ile), and salicylic acid (SA) in wild-type Arabidopsis Col-0 and mutant cop1-21 plants before and after infection with the fungal powdery mildew G. cichoracearum. Detailed Description of the Invention

[0036] The wild-type COP1 gene of Arabidopsis thaliana consists of 2,028 nucleotides, and the sequence is as shown in SEQ ID NO.1. It encodes a protein of 675 amino acids, and the sequence is as shown in SEQ ID NO.2.

[0037] Previous studies in the laboratory of the present inventors found that when the G at the 1943rd position of the CDS coding sequence of the Arabidopsis COP1 gene mutates to A (i.e., the serine at the 648th position of the encoded COP1 mutant protein mutates to asparagine), it can flower earlier under both long-day and short-day conditions, and the growth of the plants is not inhibited. This mutant gene was named cop1-21, and the corresponding encoded protein was named COP1 S648N protein. The structural schematic diagrams of the mutant gene and the mutant protein are as shown in Figure 1 and 2 (see CN116254238A). The CDS coding nucleotide sequence of cop1-21 is as shown in SEQ ID NO.3, and the amino acid sequence of the COP1 S648N protein is as shown in SEQ ID NO.4. And subsequent experiments also proved that the V596A, F646A, or S664S mutations also affect the binding to CO.

[0038] The present inventor found that Arabidopsis cop1-21 has resistance to plant EDS1-related diseases. This may be because the binding of the cop1-21 mutant to the upstream ligand that causes the accumulation of the EDS1 protein is similar to the binding of the cop1-21 mutant to CO. Therefore, cop1-21 enhances the resistance of plants to EDS1-related diseases by causing the accumulation of the EDS1 protein, and the change in the structure of the 5 Å CO-binding region does not affect the growth and development of plants. Since the V596A, F646A, or S664S mutations also affect the binding to CO, these three mutations also have the effect of causing the accumulation of the EDS1 protein, thereby enhancing the resistance of plants to EDS1-related diseases.

[0039] Based on this, the laboratory where the present inventor is located conducted a comparative analysis of the three-dimensional structures of the Arabidopsis COP1 protein and its variant protein COP1 S648N and found that within a 5 Å range centered on S648 in the COP1 protein, a small region consisting of 16 amino acids is formed. The substitution of one amino acid in this 5 Å range region changes the structure of this region, affects the interaction between COP1 and the CO protein, causes the accumulation of the CO protein, and makes Arabidopsis plants have an early-flowering phenotype, flowering under short-day or dark conditions. However, this structural change does not affect other functions of the COP1 protein. For the convenience of description, in the present invention, this 5 Å region is called the 5 Å CO-binding region. The 5 Å CO-binding region consists of 16 amino acids. Taking the Arabidopsis COP1 protein as an example, they are I373, V374, S375, S376, Y554, F595, V596, G597, F646, I647, S648, A649, V650, A662, N663, S664. Among these 16 amino acids, 10 are non-polar hydrophobic amino acids and 6 are polar neutral amino acids, 4 of which are serine. By using the Pymol software to analyze the three-dimensional structure (PBD ID: 5IGO) of the WD40 domain of the COP1 protein, it is found that 12 hydrogen bonds are formed among these 16 amino acids. Coupled with 10 non-polar hydrophobic amino acids, a stable structure maintained by hydrophobic interaction and hydrogen bonds is formed in this region, as Figures 3 - 5 shown (see CN116254238A).

[0040] Uljon et al. (Uljon et al., 2016, Structure 24, 687-696) studied in detail the three-dimensional structure of the WD40 domain of Arabidopsis thaliana COP1, which is a seven-blade β-propeller structure with an inserted loop on the bottom surface of the first blade. The seven blades are the first blade composed of sheets 1A, 1B, 1C, 1D, the second blade composed of 2A, 2B, 2C, 2D, the third blade composed of 3A, 3B, 3C, 3D, the fourth blade composed of 4A, 4B, 4C, 4D, the fifth blade composed of 5A, 5B, 5C, 5D, the sixth blade composed of 6A, 6B, 6C, 6D, and the seventh blade composed of 7A, 7B, 7C, 7D. Among them, the A sequence is located inside the three-dimensional structure, and the D sequence is located on the outer edge of the three-dimensional structure. In the primary amino acid structure, from the N-terminus to the C-terminus, they are 7D, 1A, 1B, 1C, 1D, 2A, 2B, 2C, 2D, 3A, 3B, 3C, 3D, 4A, 4B, 4C, 4D, 5A, 5B, 5C, 5D, 6A, 6B, 6C, 6D, 7A, 7B, 7C. The three-dimensional structure formed by these seven blades shows a shallow pocket at the top and a slightly deeper pocket at the bottom. Through analysis, it was found that the amino acid sites I373, V374, S375, S376, Y554, F595, V596, G597, F646, I647, S648, A649, V650, A662, N663, S664 of the present invention are respectively located in the 1A, 5A, 6A, 7A, and 7B sheet regions, that is, in the inner core of the shallow pocket of the WD40 domain, close to the sixth and seventh blades.

[0041] Since the WD40 domain of COP1 interacts with many kinds of proteins and thus degrades the bound proteins through its E3 ligase activity, such as with HY5, HYH, STO, STH, HFR1, cry1, cry2, phyA, phyB (Chunling Yi and Xingwang Deng, COP1 - from plant photomorphogenesis to mammalian tumorigenesis, Trends in Cell Biology, 2005, 15(11): 618 - 625). Therefore, the WD40 domain responsible for binding to multiple ligand proteins in the COP1 sequence is very conserved. Mutations at this domain position usually lead to structural changes and thus cause adverse negative effects. For example, Xing-Wang Deng found that many mutants of the WD40 domain are lethal, such as cop1-5, cop1-7, cop1-8, cop1-9, cop1-10, and cop1-11 (Timothy W. McNellis, Albrecht G. von arnlm, Takashi Araki, Yoshibuml Komeda, Simon Misera and Xing-Wang Deng, 1994, Genetic and Molecular analysis of an Allelic Series of cop1 Mutants Suggests Functional Roles for the Multiple Protein Domains, The Plant Cell, 6: 487 - 500), and it was also found that in the lethal mutants cop1-8 and cop1-9, the COP1-8 and COP1-9 variant proteins cannot interact with HY5, STH, and STO (Magnus Holm, Christian S. Hardtke, Rachelle Gaudet and Xing-Wang Deng, 2001, Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1, The EMBO Journal, 20(1&2): 118 - 127).

[0042] According to research, many COP1 binding ligands, such as HY5, STO, STH, etc., bind to the core of the WD40 domain of COP1 near the sides of the 3rd, 4th, and 5th blades, and interact through ionic bonds (Uljon et al. 2016, Structure 24, 687 - 696). According to the analysis, mutations in other WD40 domains lead to significant changes in the three-dimensional structure of the COP1 protein, affecting the binding of some ligands, resulting in lethal phenotypes or other phenotypic defects. In the present invention, the local structure composed of 16 amino acids centered on S648 is very stable due to hydrophobic interactions and hydrogen bond interactions. The mutation of one amino acid only produces a small local change in the three-dimensional structure, which only affects the core inside the shallow pocket near the sixth and seventh blades, and the structure on the other side of the core should not be affected. Therefore, only the trait of disease resistance due to the accumulation of the EDS1 protein appears, and no other growth and development defects, such as dwarf plants, etc., will occur.

[0043] As is well known, there are 20 kinds of amino acids that make up organisms. According to the properties of the amino acid side chains, amino acids are divided into 4 categories: ① Amino acids with non-polar, hydrophobic side chains, including alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), glycine (Gly, G), methionine (Met, M), tryptophan (Trp, W), and phenylalanine (Phe, F); ② Amino acids with polar, neutral side chains, including glutamine (Gln, Q), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), asparagine (Asn, N), and tyrosine (Tyr, Y); ③ Amino acids with polar, acidic side chains, including aspartic acid (Asp, D) and glutamic acid (Glu, E); ④ Amino acids with polar, basic side chains, including lysine (Lys, K), arginine (Arg, R), and histidine (His, H). In the present invention, the terms non-polar hydrophobic amino acids and polar neutral amino acids refer to the amino acids in the above-mentioned first and second categories. In the present invention, the term substitution of amino acids of the same type means substituting an amino acid in one of the above four categories of amino acid definitions with an amino acid of the same category as it. In the present invention, in the 5 Å CO binding region formed by the 16 amino acids, any type of amino acid substitution for one amino acid can achieve the technical effect of only affecting this local structure and thus only affecting the accumulation of EDS1. Therefore, any type of amino acid substitution is an embodiment of the present invention. Since this local region includes 10 non-polar hydrophobic amino acids and 6 polar neutral amino acids, those skilled in the art know that substituting with amino acids of the same type has a relatively small impact on the local three-dimensional structure. Therefore, preferably, the present invention is implemented by substituting non-polar hydrophobic amino acids or polar neutral amino acids with amino acids of the same type.

[0044] To study the conservation of the amino acids in the above-mentioned 5-angstrom CO-binding region in angiosperms, the laboratory where the present inventors are located has previously selected some plants in angiosperms for sequence alignment. These plants include Arabidopsis thaliana (GenBank: GenBank: ABF57293.1), Brassica napus (GenBank: CAF2096033.1), and Raphanus sativus (NCBI Reference Sequence: XP_018441855.1) in the Brassicaceae family, Fragaria × ananassa (GenBank: API61819.1), Rosa chinensis (NCBI Reference Sequence: XP_024173448.1), Malus domestica (NCBI Reference Sequence: XP_028951334.1), and Prunus persica (NCBI Reference Sequence: XP_007210464.2) in the Rosaceae family, Glycine max (NCBI Reference Sequence: XP_003545597.1) and Arachis hypogaea (NCBI Reference Sequence: XP_025609023.1) in the Fabaceae family, Triticum aestivum (GenBank: KAF7091270.1), Hordeum vulgare (GenBank: KAE8814141.1), Zea mays (NCBI Reference Sequence: XP_008677082.1), Sorghum bicolor (NCBI Reference Sequence: XP_021315078.1), Oryza sativa subsp. japonica (NCBI Reference Sequence: XP_015627602.1), and Oryza sativa subsp. indica (GenBank: EEC74080.1) in the Poaceae family, Nicotiana tabacum (NCBI Reference Sequence: XP_016503261.1), Solanum lycopersicum (GenBank: AAC98912.1), and Solanum tuberosum (NCBI Reference Sequence: XP_006351972.1) in the Solanaceae family, Daucus carota (NCBI Reference Sequence: XP_017228304.1) in the Apiaceae family, Helianthus annuus (NCBI Reference Sequence: XP_021989424.1) in the Asteraceae family, Gossypium hirsutum (NCBI Reference Sequence: XP_016710447.2) in the Malvaceae family, and Cucumis sativus (NCBI Reference Sequence: XP_004143233.1) in the Cucurbitaceae family. The corresponding GenBank accession numbers or NCBI reference sequence numbers are given behind these plants, which can facilitate the query of the corresponding sequence information from the publicly available databases.The comparative software used was the software ClustalW. Through alignment, it was found that the WD40 domain of COP1 in many angiosperms (amino acids 349 to 675 in Arabidopsis thaliana as an example) is relatively conserved compared to other domains. Although there are 185 identical amino acids among the 327 amino acids in the WD40 domain, with an identity of 56.6%, a quite large part of the amino acids in the sheet region forming the seven paddles are completely identical, and the 16 amino acids in the 5 Å CO-binding region are completely identical, as Figure 6 shown (see CN116254238A).

[0045] The laboratory where the present inventors are located further conducted the same sequence alignment on cruciferous plants, which are: Arabidopsis thaliana of the genus Arabidopsis (GenBank: ABF57293.1), Arabis alpina of the genus Arabis (GenBank: KFK31097.1), Thellungiella salsuginea of the genus Thellungiella (NCBI Reference Sequence: XP_006410433.1), Camelina sativa of the genus Camelina (NCBI Reference Sequence: XP_010509945.1), Capsella bursa-pastoris of the genus Capsella (NCBI ReferenceSequence: XP_023640041.1), Raphanus sativus of the genus Raphanus (NCBI Reference Sequence: XP_018441855.1), Sinapis alba of the genus Sinapis (GenBank: KAF8049993.1), Brassica oleracea of the genus Brassica (NCBI ReferenceSequence:

[0046] XP_013637215.1), Brassica oleracea var. botrytis (GenBank: KAF3554934.1), Brassica rapa (GenBank:

[0047] AEE81754.1) and Brassica napus (GenBank: CAF2096033.1). The corresponding GenBank accession numbers or NCBI reference sequence numbers are given behind these plants, and the corresponding sequence information can be conveniently queried from the publicly available database. Through sequence comparison, it was found that the sequence identity of the WD40 domain of COP1 in cruciferous plants (amino acids 349 to 675 in Arabidopsis thaliana as an example) is 95.7%. Among the 327 amino acids, the amino acids at 14 positions are different in each cruciferous plant. Among them, 13 amino acids have substitutions, and most of them are substitutions of the same type of amino acids. Another difference is that the A corresponding to position 408 in Arabidopsis thaliana is missing in Arabis alpina. And the 16 amino acids in the 5 Å CO-binding region are completely conserved in these plants, with an identity of 100%, as Figure 7as shown (see CN116254238A).

[0048] For the above-mentioned other angiosperm plants, due to their 100% identity with the 5 Å CO-binding region of the Arabidopsis thaliana COP1 protein and the relatively high conservation of the WD40 domain in Brassicaceae plants, combined with the existing plant disease resistance signaling pathway theory and the result that the following COP1 variants produce disease resistance through the accumulation of EDS1 protein, it is believed that similar mutations of the COP1 protein in other angiosperm plants also have the same disease resistance effect, especially through the COP1-EDS1-SA-regulated disease resistance pathway. Because the COP1 in these plants has a sequence identity of 80%-90% with Arabidopsis thaliana COP1, especially the WD40 region has a highly conserved identity. Moreover, the 16 amino acids in the 5 Å CO-binding region described in the present invention are 100% identical in different plant species, and the amino acid types are also very conserved. Most of this region is non-polar amino acids and is uncharged. Therefore, they form a conserved domain through local hydrophobic interactions. Therefore, the three-dimensional configuration of this domain is of great significance. Since the 16 amino acids are still the same in the above-mentioned wide range of angiosperm species, it can be seen that the domain of COP1 is the same. Therefore, by replacing one amino acid at the equivalent position of the 16 amino acids in the 5 Å CO-binding region centered on Arabidopsis thaliana S648 in all other species, the disease resistance effect of these species can also be achieved. Therefore, the corresponding COP1 variant genes or variant proteins in these species are also technical solutions that can achieve the technical effects of the present invention and are also within the protection scope of the present invention.

[0049] Accordingly, one aspect of the present invention relates to the use of a COP1 variant protein for combating plant diseases, wherein the COP1 variant protein is a variant of the COP1 protein of angiosperm plants, and one amino acid at positions I373, V374, S375, S376, Y554, F595, V596, G597, F646, I647, S648, A649, V650, A662, N663, S664 in the amino acid sequence of SEQ ID NO: 2 is replaced, or based on the protein sequence alignment with SEQ ID NO: 2, one amino acid corresponding to positions I373, V374, S375, S376, Y554, F595, V596, G597, F646, I647, S648, A649, V650, A662, N663, S664 in other angiosperm COP1 proteins is replaced; the coding nucleic acid molecule is any form of nucleic acid molecule encoding the above COP1 variant protein, including but not limited to DNA, cDNA, mRNA, etc. The above-mentioned other angiosperm plants refer to angiosperm plants other than Arabidopsis thaliana, including the above-mentioned Arabis alpina, Thellungiella halophila, Camelina sativa, Capsella bursa-pastoris, Raphanus sativus, Sinapis alba, Brassica oleracea, Brassica oleracea var. botrytis, Brassica rapa, Brassica napus, Fragaria × ananassa, Rosa chinensis, Malus domestica, Prunus persica, Glycine max, Arachis hypogaea, Triticum aestivum, Hordeum vulgare, Zea mays, Sorghum bicolor, Oryza sativa subsp. japonica, Oryza sativa subsp. indica, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum, Daucus carota, Helianthus annuus, Gossypium hirsutum, Cucumis sativus.

[0050] Preferably, the above amino acid substitutions are made with polar neutral amino acids or non-polar hydrophobic amino acids. Preferably, the serine at position S648 is replaced with an amino acid of the same type, for example, serine is replaced with asparagine, glutamine, tyrosine, threonine, cysteine or glycine. Most preferably, the serine at position 648 is replaced with asparagine.

[0051] In another aspect, the present invention provides the use of a nucleic acid molecule encoding the above COP1 variant protein for combating plant diseases, wherein the nucleic acid molecule encodes the above COP1 variant protein. As is well known, due to the degeneracy of biological codons, that is, one amino acid can be encoded by one or more codons, there are very many nucleic acid molecules encoding the COP1 variant protein of the present invention and it is impossible to list them all. As long as the nucleic acid molecule can express the COP1 variant protein of the present invention, the property of combating plant diseases can be achieved. Therefore, the nucleic acid molecules encoding the COP1 variant protein that can endow plants with disease resistance characteristics in the present invention are not limited to the Arabidopsis thaliana nucleotide sequence SEQ ID NO: 3. And the design and nucleic acid molecule synthesis based on the COP1 variant protein of the present invention are well known in the art.

[0052] As described above, different technical means are used to enable plants to express the COP1 variant protein of the present invention. By promoting the accumulation of EDS1 in plants, the plants exhibit disease resistance characteristics. The plant varieties thus produced can have very important economic value. And as mentioned above, in the plants analyzed, especially in cruciferous plants, the WD sequence identity of COP1 is very high. Therefore, for each plant, the heterologous COP1 variant protein from other species also has the effect of making the plant disease-resistant, especially when the plant species are closely related, such as different species in the same family, and further, different species in the same genus. Therefore, such heterologous applications are also within the scope of protection of the present invention.

[0053] In the present invention, plants with plant disease resistance characteristics are propagated and cultivated by in vitro culturing of cells. Specifically, the COP1 variant gene or expression vector of the present invention is introduced into plant cells, and then the plant cells are cultivated into plants. The introduced variant gene is recombinantly integrated into the plant genome, enabling the plant cells to express the COP1 variant protein of the present invention. The expression vectors, introduction reagents or methods, gene recombination integration, and in vitro culturing techniques for this purpose are well known in the art. In addition, plants with early flowering characteristics can also be cultivated by traditional hybridization methods. Specifically, a plant expressing the above-mentioned COP1 variant protein is hybridized with a plant not expressing the above-mentioned COP1 variant protein, and then the progeny plants expressing the above-mentioned COP1 variant protein are screened out.

[0054] For the purpose of this application of the present invention only, in the present invention, the term "plant disease" is defined as any type of disease that can generate resistance through the signal pathway with an increase in the content of salicylic acid (SA). The term "EDS1-related disease" refers to any type of plant disease that can generate resistance through the accumulation of EDS1 protein and the downstream signal pathway with an increase in the content of salicylic acid (SA). EDS1-related diseases include but are not limited to:

[0055] 1. Powdery mildew caused by the infection of powdery mildew fungi

[0056] Powdery mildew, such as but not limited to wheat powdery mildew, TaEDS1 has the highest expression level in wheat leaves and is induced by Blumeria graminis f. sp. tritici (Bgt) and salicylic acid (SA), but is not regulated by jasmonic acid. After down-regulating the expression of TaEDS1 by BSMV-VIGS, Shangeda and Xiaobaidongmai lost their resistance to Bgt. In the leaf cells of the powdery mildew-sensitive cultivar Chancellor with transient overexpression of the TaEDS1 gene, the infection index of Bgt decreased significantly; expressing the TaEDS1 gene in the Arabidopsis mutant eds1-1 complemented its sensitive phenotype to Golovinomyces cichoracearum (Gc, UJCSC1). The above experimental results indicate that the TaEDS1 protein may positively regulate the resistance response of wheat to powdery mildew through the SA signaling pathway (Chen, G., Wei, B., Li, G. et al. TaEDS1 genes positively regulate resistance to powdery mildew in wheat. Plant Mol Biol 96, 607–625 (2018)).

[0057] 2. Downy mildew caused by fungal infection of Peronospora spp.

[0058] Scientists have discovered a variety of proteins that can help plants enhance their own immunity and improve their resistance to downy mildew. Many important genes encode R proteins, such as SGT1b, RAR1, and EDS1. These genes can enhance the resistance of crops to fungi and other pathogens through their interaction with each other (Tian, S., et al., Ectopic Expression of Grapevine Gene VaRGA1 in Arabidopsis Improves Resistance to Downy Mildew and Pseudomonas syringae pv. tomato DC3000 But Increases Susceptibility to Botrytis cinerea. Int J Mol Sci, 2019. 21(1); Ali Ramuli Máquina Riquicho, Analysis of the expression of related resistance genes in non-heading Chinese cabbage after treatment with downy mildew pathogen, Master's thesis of Nanjing Agricultural University, 2015).

[0059] 3. Capsicum blight caused by fungal infection of Phytophthora spp.

[0060] Such Phytophthora diseases include, for example but not limited to, Phytophthora capsici, whose effector protein PcAvh103 suppresses plant immunity by specifically interacting with the lipase domain of plant EDS1 and disrupting the EDS1-PAD4 immune signaling pathway (Li, Q., et al., A Phytophthora capsici effector suppresses plant immunity via interaction with EDS1. Mol Plant Pathol, 2020. 21(4): p. 502-511; Parra, G. and J. Ristaino, Insensitivity to Ridomil Gold (Mefenoxam) Found Among Field Isolates of Phytophthora capsici Causing Phytophthora Blight on Bell Pepper in North Carolina and New Jersey. Plant Dis, 1998. 82(6): p. 711).

[0061] 4. Alternaria leaf spot caused by the infection of the fungus Alternaria alternata

[0062] Such Alternaria leaf spot includes, for example but not limited to, Chrysanthemum (Chrysanthemum morifolium) black spot disease (CBS). It has been found that in the early stage of pathogen infection, calcium signaling and EDS1-mediated immune responses are activated; as the lesions form, more plant immune responses come into play, especially ethylene-signaling-mediated immune responses (Xin, J., et al., CmMLO17 and its partner CmKIC potentially support Alternaria alternata growth in Chrysanthemum morifolium. Hortic Res, 2021. 8(1): p. 101).

[0063] 5. Bacterial wilt caused by the infection of the bacterium Ralstonia solanacearum

[0064] This type of bacterial wilt, such as but not limited to eggplant bacterial wilt, with the Latin name: Ralstonia pseudo solanacearum. Silencing of the SmEDS1 gene can significantly affect the expression levels of other signaling genes. Among them, the expression levels of MAPK6 and RAR1 are up-regulated, while the expression levels of genes such as MAPK3, SIPK, PAD4, SGT1, TGA, EDR1, NPR1, ICS1, GLUA, IL1, and HSP90 are down-regulated. The expression levels of EBF2 and AC05 are not regulated by SmEDS1. This result indicates that EDS1 plays an important role in regulating eggplant resistance to bacterial wilt (Li Ke, Xiao Xi'ou, Lin Wenqiu, Li Wei, Lü Lingling, Cao Bihao, EDS1 positively regulates the eggplant response to bacterial wilt, Chinese Journal of Tropical Crops 2018, 39(2): 332 - 337).

[0065] 6. The following diseases caused by Pseudomonas syringae:

[0066] Tomato bacterial leaf spot, with the pathogen name Pseudomonas syringae pv. tomato, and the host plant being Tomato;

[0067] Wildfire of tobacco, with the pathogen name Pseudomonas syringae pv. tabaci, and the host plant being Tobacco of the Solanaceae family;

[0068] Snapdragon blight, with the pathogen name Pseudomonas syringae pv. antirrhini, and the host plant being Snapdragon of the Scrophulariaceae family;

[0069] Bacterial leaf spot, with the pathogen name Pseudomonas syringae pv. apii, and the host plant being Celery of the Apiaceae family;

[0070] Bacterial blight of wheat, the pathogen is Pseudomonas syringae pv. atrofaciens, and the host plants are wheat and other cereal plants;

[0071] Berberis bacterial leaf spot, the pathogen is Pseudomonas syringae pv. berberidis, and the host plants are Japanese barberry Berberis and other plants of the genus Berberis;

[0072] Hemp bacterial blight, the pathogen is Pseudomonas syringae pv. cannabina, and the host plants are Cannabis sativa of Moraceae, Glycine max of Leguminosae, and Vicia sativa;

[0073] Bacterial blight, the pathogen is Pseudomonas syringae pv. ciccaronei, and the host plant is Algaroba of Leguminosae;

[0074] Halo blight, the pathogen is Pseudomonas syringae pv. coronafacien, and the host plants are oats and maize;

[0075] Delphinium black spot, the pathogen is Pseudomonas syringae pv. delphinii, and the host plants are Delphinium plants of Ranunculaceae;

[0076] Bacterial leaf spot, the pathogen is Pseudomonas syringae pv. helianthi, and the host plant is sunflower of Compositae;

[0077] Bacterial blight of soybean, the pathogen is Pseudomonas syringae pv. glycinea, and the host plant is soybean in Leguminosae;

[0078] Cherry, plum canker, the pathogen is Pseudomonas syringae pv. morsprunorum, and the host plant is plants in Prunus of Rosaceae;

[0079] Melon bacterial spot disease, the pathogen is Pseudomonas syringae pv. lachrymans, and the host plants are cucumber, watermelon, muskmelon, etc. in Cucurbitaceae;

[0080] Coffee faint, the pathogen is Pseudomonas syringae pv. garcae, and the host plant is coffee in Rubiaceae;

[0081] Bacterial black rot, the pathogen is Pseudomonas syringae pv. maculicola, and the host plant is Cruciferae;

[0082] Myrica rubra cancer granulomatosis, the pathogen is Pseudomonas syringae pv. myricae, and the host plant is Myrica rubra;

[0083] Millet brown streak, the pathogen is Pseudomonas syringae pv. panici, and the host plant is millet grass in Gramineae;

[0084] Apple herpes, the pathogen is Pseudomonas syringae pv. papulans, and the host plants are apple and pear of Rosaceae;

[0085] Peach bacterial blight, the pathogen is Pseudomonas syringae pv. persicae, and the host plant is peach of Rosaceae;

[0086] Halo blight of bean, the pathogen is Pseudomonas syringae pv. phaseolicola, and the host plant is pea bean of Leguminosae;

[0087] Pea bacterial blight, the pathogen is Pseudomonas syringae pv. pisi, and the host plant is pea of Leguminosae;

[0088] Bacterial leaf spot, the pathogen is Pseudomonas syringae pv. sesami, and the host plant is Pedaliaceae flax;

[0089] Heaf stripe and seedling blight of oat, the pathogen is Pseudomonas syringae pv. striafaciens, and the host plants are oats and barley of Gramineae;

[0090] Clove blight, the pathogen is Pseudomonas syringae pv. syringae, and the host plants include clove of Oleaceae, stone fruit trees of Rosaceae, etc.;

[0091] Tagetes bacterial leaf spot, the pathogen is Pseudomonas syringae pv. tagetis, and the host plant is Compositae Tagetes;

[0092] Camellia blight, the pathogen is Pseudomonas syringae pv. theae, and the host plant is Camellia of Theaceae;

[0093] Bacterial canker of kiwifruit, the pathogen is Pseudomonas syringae pv. actinidiae, and the host plant is Actinidia;

[0094] Rotten disease, the pathogen is Pseudomonas syringae pv. mori, and the host plant is Moraceae;

[0095] Platycodon leaf spot disease, the pathogen is Pseudomonas syringae pv. platicodon pv. nov, and the host plant is Campanulaceae (Wang Dandan, Wang Qingming, Research progress on the molecular biology of Pseudomonas syringae, Acta Agriculturae Boreali-occidentalis Sinica, 2017, 26(4): 487-496).

[0096] When plants are infected by Pst DC3000, EDS1 will rapidly promote the biosynthesis of salicylic acid SA and the expression of resistance-related genes to initiate the plant's defense response. At the same time, pathogen infection will stabilize the DELLA proteins RGA and RGL3, restricting plant growth in a partially EDS1-dependent manner, thus benefiting the plant's resistance to pathogens (Li, Y., et al., DELLA and EDS1 Form a Feedback Regulatory Module to Fine-Tune Plant Growth-Defense Tradeoff in Arabidopsis. Mol Plant, 2019. 12(11): p. 1485-1498).

[0097] Research by Gangappa et al. has shown that since DET1 and COP1 negatively regulate immunity, they are crucial for the immune regulation of photoperiod and temperature and are regulated by the transcription factor PIF4, indicating that the DET1 / COP1-PIF4 module serves as a central hub for controlling growth and immunity in response to seasonal signals (Gangappa, S.N. and S.V. Kumar, DET1 and HY5 Control PIF4-Mediated Thermosensory Elongation Growth through Distinct Mechanisms. Cell Rep, 2017. 18(2): p. 344-351).

[0098] Since the COP1 mutant of the present invention achieves resistance to powdery mildew through the accumulation of EDS1 protein and the elevation of downstream SA levels, mutants similar to the COP1 mutant of the present invention in other angiosperm plants will also be resistant to the above-mentioned plant diseases, especially EDS1-related diseases, through the accumulation of EDS1 protein and the elevation of downstream SA levels. Therefore, the above-mentioned EDS1-related diseases are within the protection scope of the present invention.

[0099] The present invention will be further described below in conjunction with specific embodiments, but these embodiments in no way limit the protection scope of the present invention. The experimental methods and techniques used in the embodiments are conventional techniques in the art unless otherwise specified, and the reagents and materials used are conventional reagents and materials that can be commercially purchased unless otherwise specified. Embodiments

[0100] Example 1 Experimental Materials and Experimental Reagents

[0101] For the establishment of the wild-type Columbia-0 (Col-0) and the mutant cop1-21 of Arabidopsis thaliana used in this invention application, refer to Chinese Patent Application Publication No. CN116254238A. For the establishment of the mutants cop1-4 and cop1-6 as controls, refer to Chinese Patent Application Publication No. CN1329670A, as well as the journal articles McNellis TW, von Arnim AG, Araki T, Komeda Y, Miséra S, Deng XW. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains. Plant Cell. 1994, 6(4):487-500 and Ang LH, Deng XW. Regulatory hierarchy of photomorphogenic loci: allele-specific and light-dependent interaction between the HY5 and COP1 loci. Plant Cell. 1994, (5):613-28. The COP1 overexpression plant 35S::COP1-GFP was prepared as follows: The CDS sequence of COP1 (SEQ ID NO.1) was inserted into the pCambia1300-35S-EGFP plant expression vector through the restriction enzyme site ApaI to construct an expression vector, and then the COP1 overexpression plant 35S::COP1-GFP was obtained by transfection into Arabidopsis thaliana and screening with Kana. The eds1-2 plants were prepared as described by Jane E. Parker et al. (Jane E. Parker et al., 1996, Characterization of eds1, a Mutation in Arabidopsis Suppressing Resistance to Peronospora parasitica Specified by Several Different RPP Genes, The Plant Cell, 8:2033-2046).

[0102] The plants were grown under short-day light (150 μE / m2 s) (9 h light / 15 h dark) at a temperature of 22 °C and a relative humidity of 65%.

[0103] The experimental reagents used in this invention application are prepared as follows:

[0104] 1. The trypan blue staining solution required for trypan blue staining is prepared as shown in Table 1 and stored at 4°C. The decolorizing solution is prepared as shown in Table 2, mixed well and stored at 4°C.

[0105] Table 1 Trypan blue staining solution

[0106]

[0107] Table 2 Decolorizing solution

[0108]

[0109] 2. Solutions I, II, and III required for plasmid extraction are prepared as shown in Tables 3 - 5 below. Solution I and III are stored at 4°C, and solution II is stored at room temperature.

[0110] Table 3 Solution I

[0111]

[0112] Table 4 Solution II

[0113]

[0114] Table 5 Solution III

[0115]

[0116] 3. The protein extraction buffer for protein extraction is prepared as shown in Table 6 below.

[0117] Table 6 Protein extraction buffer

[0118]

[0119]

[0120] 4. The reagents used for transient protoplast transformation are prepared as shown in Tables 7 - 12 below.

[0121] Table 7 0.8mol / L mannitol

[0122]

[0123] Table 8 200mmol / L MES

[0124]

[0125] Adjust the pH to 5.7 with KOH and make up the volume to 100 mL with dd H2O.

[0126] Table 9 Enzyme digestion solution

[0127]

[0128] Make up to 40 mL with dd H2O and store at 4°C.

[0129] Table 10W5

[0130]

[0131] Add 400 mL of dd H2O, mix well, make up to 500 mL and store at 4°C.

[0132] Table 11MMG

[0133]

[0134] Store at -80°C.

[0135] Table 12 45% PEG

[0136]

[0137] Add dd H2O to make up to 40 mL and store at room temperature.

[0138] 5. Solutions for protein purification, immunoprecipitation and immunoblotting are prepared as shown in Tables 13 - 18 below.

[0139] Table 13 1 mol / L Tris-HCl (pH = 6.8 / 7.5 / 8.0)

[0140]

[0141] Mix well, adjust the pH to 6.8 / 7.5 / 8.0 with concentrated hydrochloric acid, and make up to 1 L with dd H2O.

[0142] Table 14 3 mol / L Sodium Acetate

[0143]

[0144] Adjust the pH to 5.2 with glacial acetic acid, make up to 100 mL with dd H2O, sterilize by autoclaving and store at room temperature.

[0145] Table 15 1 mol / L DTT

[0146]

[0147] After complete dissolution, filter through a 0.22 μm filter to sterilize, aliquot and store at -20°C.

[0148] Table 16 Wash Buffer

[0149]

[0150]

[0151] Make up the volume to 100 mL with dd H2O and store at 4°C.

[0152] Table 17 EWB

[0153]

[0154] Store at 4°C.

[0155] Table 18 2× Loading Buffer

[0156]

[0157] After aliquoting, store at -20°C.

[0158] Example 2: Growth and Developmental Phenotypes of cop1-21 Mutant Plants

[0159] In this experiment, the wild-type Col-0 was used as an essential reference for Arabidopsis experiments. In the cop1-21 mutant, the 1943rd nucleotide G in the COP1 gene was mutated to A. This mutant was named cop1-21. Its CDS-encoding nucleotide sequence is shown in SEQ ID NO.3, and the encoded protein sequence is shown in SEQ ID NO.4. Among the cop1-4 and cop1-6 mutant controls, the cop1-4 mutant lacks the entire WD40 domain, and the cop1-6 mutant has an in-frame insertion before the WD40 domain.

[0160] First, the wild-type Col-0 and the mutants cop1-21, cop1-4, and cop1-6 were cultured under short-day conditions until they reached 4 weeks of age. After taking pictures, the above-ground parts were cut off along the soil surface, and their weights were measured using an analytical balance. Thirty samples of each material were measured, and the fresh weights of the wild-type Col-0 and the cop1-21, cop1-4, and cop1-6 mutants were calculated using this method. The experimental results are shown in Figure 8 . As Figure 8 shown in A, the mutation at the S648N site does not affect the normal development of plants. By comparing the plant type size and the calculated fresh weight, the cop1-21 mutant with the S648N site mutation even had a significant increase in fresh weight compared to Col-0. Compared with the wild-type Col-0, the plant types of the cop1-4 and cop1-6 mutants were very short, and their fresh weights were significantly reduced, indicating that the cop1-4 and cop1-6 mutants affected the normal growth and development of the plants.

[0161] Example 3: Powdery Mildew Resistance Phenotypes of cop1-21 Mutant Plants

[0162] The preparation of powdery mildew for inoculation is as follows: Powdery mildew cultured on eds1-2 plants of susceptible materials for about 7 days is blown onto experimental materials with a hair dryer for about 30 s through an inoculation device, left standing for 30 min and then transferred to an incubator, and the disease-resistant phenotype is observed at 7 dpi.

[0163] Wild-type Col-0 and mutant cop1-21, cop1-4, cop1-6 and COP1 overexpression plants 35S::COP1-GFP were cultured under short-day conditions until they were 4 weeks old, and then inoculated with a large amount of powdery mildew (G. cichoracearum) (blown with a hair dryer for about 30 s). Leaves of each plant were photographed 7 days after inoculation. At the same time, a part of the plants were sampled 5 days after inoculation with a small amount of powdery mildew (blown with a hair dryer for about 5 s), an appropriate amount of trypan blue was added for staining, placed in an oven at 65 °C overnight, the trypan blue staining solution was poured out, and an appropriate amount of decolorizing solution was added for decolorization for 12 h. After rinsing with water, they were stored in 50% glycerol. After making slides, the number of conidiophores was counted under a microscope, and the disease-resistant phenotype was determined according to the number of conidiophores of a single spore. Compared with Col-0, more conidiophores indicate susceptibility, and fewer conidiophores indicate disease resistance.

[0164] Photos of the leaf infection conditions of wild-type and mutant plants after inoculation with a large amount of powdery mildew are as Figure 9 shown. Seven days after inoculation with a large amount of powdery mildew, there was a large amount of white powder on the surface of Col-0 leaves, while there was no visible powder on the surface of cop1-21 leaves.

[0165] Photos of trypan blue staining of leaves of wild-type Col-0 and mutants cop1-21, cop1-4, cop1-6 and 35S::COP1-GFP Arabidopsis thaliana after quantitative inoculation with powdery mildew are as Figure 10 shown. Compared with Col-0, Arabidopsis thaliana cop1-21, cop1-4, cop1-6 had fewer conidiophores, and 35S::COP1-GFP had more conidiophores. The quantification diagram of conidiophores calculated from the Figure 10 trypan blue staining is as Figure 11 shown, where different lowercase letters on the violin plot represent significant differences. It can be seen that compared with Col-0 (the number of conidiophores was 54.9), Arabidopsis thaliana cop1-21, cop1-4, cop1-6 had significantly fewer conidiophores, 22.6, 12.4 and 29.3, and the conidiophores of COP1 overexpression Arabidopsis thaliana were significantly more, 80.6.

[0166] Example 4: COP1 S648N Interaction experiment with EDS1 protein

[0167] To explore the effect of the S648N mutation on the interaction between COP1 and EDS1 proteins, the inventors used the co-immunoprecipitation method (Co-Immunoprecipitation, Co-IP) to experiment on the interaction between COP1-EDS1 and COP1 S648N -EDS1.

[0168] Specifically, using wild-type Col-0 or cop1-21 seedlings as materials, total RNA was extracted by the Trizol method (Invitrogen), and cDNA was reverse transcribed (HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper), #R312, Vazyme). Using the cDNA of Col-0 as a template, the full-length EDS1 coding sequence was PCR amplified with primer 1: 5’-atggtctcaATGGCGTTTGAAGCTCTTAC-3’ (SEQ ID NO.5) and primer 2: 5’-atggtctcacgaaGTATCTGTTATTTCATCCATCATATAGTC-3’ (SEQ ID NO.6). Using the cDNA of Col-0 and cop1-21 as templates respectively, the full-length wild-type COP1 and COP1S648N coding sequences were PCR amplified with primer 3: 5’-ggggacaagtttgtacaaaaaagcaggctctATGGAAGAGATTTCGACGGATC-3’ (SEQ ID NO.7) and primer 4: 5’-ggggaccactttgtacaagaaagctgggtcCGCAGCGAGTACCAGAAC-3’ (SEQ ID NO.8). The pICH-6×HA-EDS1 and pENSG-GFP-COP1 or pENSG-GFP-COP1S648N vectors were created by Golden Gate and Gateway cloning methods (GatewayTM LR ClonaseTMII Enzyme Mix, #11791, Invitrogen).

[0169] Protoplasts were prepared according to the method reported in the literature (Yoo et al., 2007, Nat Protoc(2):1565-1572). Then, the plasmid combinations YFP, YFP-COP1 or YFP-COP1 S648N and the EDS1-HA combination were transferred into protoplasts via the PEG-mediated method. The specific operation steps for the transient expression of Arabidopsis protoplasts are as follows:

[0170] 1. Cut 4-week-old Arabidopsis leaves into 1-mm-wide strips and place them in a 50-mL beaker containing enzyme solution;

[0171] 2. Evacuate the air, 5 minutes each time, for a total of two times;

[0172] 3. Wrap with tin foil paper and place in a shaker at 60 r / min for enzymatic digestion at room temperature for 2.5 h;

[0173] 4. Filter through a 100-mesh cell sieve into a 50-mL centrifuge tube, make up the volume to 40 mL with W5, and dispense into 20 mL / tube;

[0174] 5. Use a refrigerated balance centrifuge, 100 G, adjust the up and down lifting rate to 3, centrifuge at 4°C for 2 minutes, then discard the supernatant, and repeat 3 times;

[0175] 6. Add 15 mL of W5 and let it settle naturally on ice for 30 minutes. During this period, detect the quality of protoplasts through a microscope;

[0176] 7. Centrifuge at 4°C, 100 G for 1 minute, remove the supernatant, and add an appropriate amount of pre-cooled MMG and shake well;

[0177] 8. Add 100 μg of plasmid to a 2-mL centrifuge tube and add 2 mL of protoplasts and mix well;

[0178] 9. Add 2.2 mL of 45% PEG, mix well, and let stand for 10 minutes;

[0179] 10. Add 10 mL of W5, mix well, centrifuge at 4°C, 200 G for 2 minutes, and repeat 2 times;

[0180] 11. Incubate overnight under weak light.

[0181] The transformed protoplasts are incubated overnight under weak light at room temperature and then captured with GFP agarose beads (Chromotek, gtma-20) complex. The GFP agarose beads are added with an equal volume of 2×SDS-PAGE loading buffer, heated for denaturation, and then subjected to SDS-PAGE electrophoresis, membrane transfer, Western analysis, chemiluminescence color development and imaging. The specific operation steps of protein extraction, immunoprecipitation and immunoblotting are as follows:

[0182] 1. Centrifuge the overnight-cultured Arabidopsis protoplasts at 25°C, 200 G for 2 minutes;

[0183] 2. After removing the supernatant, add 1 mL of EWB and transfer it to a pre-cooled 1.5-mL centrifuge tube;

[0184] 3. Vortex and break for 30 s, and repeat 1 time;

[0185] 4. Centrifuge at 4°C, 12000 r / min for 15 minutes. If it is not clear, transfer the supernatant to a new centrifuge tube and centrifuge again for 5 minutes;

[0186] 5. Transfer the supernatant to a new centrifuge tube and take 50 μL as input;

[0187] 6. Take 15 μL of GFP agarose into a 1.5 mL centrifuge tube;

[0188] 7. Add 1 mL of washing buffer, place on ice for 1 min, centrifuge at 4 °C and 1000 G for 1 min, discard the supernatant, and repeat 3 times;

[0189] 8. Mix the plant protein supernatant with the washed GFP agarose and bind by rotation at 4 °C for 2 - 3 h;

[0190] 9. Centrifuge and discard the supernatant;

[0191] 10. Add 1 mL of WB, centrifuge at 4 °C and 1000 G for 1 min, discard the supernatant, and repeat 5 times. For the last time, aspirate the residual supernatant with a 1 mL syringe;

[0192] 11. Add 60 μL of 2× loading buffer and boil in a water bath for 10 min;

[0193] 12. Centrifuge at 10000 r / min for 2 min and transfer the supernatant to a new centrifuge tube for standby;

[0194] 13. Separate the proteins on a 12% SDS-PAGE gel (see the TetraCell user manual of Bio-Rad), transfer the membrane (see the Mini Electrophoretic Transfer Cell user manual of Bio-Rad). Use anti-HA (#11867423001, Roche) and anti-GFP (M20004, Abmart) as primary antibodies respectively for Western blot analysis. After HRP chemiluminescence color development (XD343079, Thermo), image with the Bio-Rad fully automatic chemiluminescence image analysis system.

[0195] In Western analysis, anti-HA antibody (#11867423001, Roche) was used to detect EDS1-HA, and anti-GFP antibody (M20004, Abmart) was used to detect GFP-COP1 or GFP-COP1 S648N .

[0196] COP1 S648N The Western blot analysis results of the co-immunoprecipitation of COP1 Figure 12 with EDS1 are shown in

[0197] . The results show that a large amount of EDS1 protein can co-precipitate with COP1 protein and COP1 variant proteins.Through the detection of endogenous proteins, we found that compared with the wild-type Col-0, the accumulation of endogenous EDS1 protein increased in the cop1-21 mutant and was induced by Blumeria graminis f. sp. hordei. Through Co-ip experiments, we found that there was an interaction between COP1 and EDS1, but the S648N mutation had no effect on this interaction. We speculate that the S648N mutation does not affect the interaction between COP1 and EDS1, but affects its subsequent processes, such as ubiquitination, etc. The specific mechanism is not clear yet.

[0198] Example 4: Experiment on the abundance of EDS1 protein

[0199] Since COP1 S648N interacts with the EDS1 protein and the possible S648N of COP1 may lead to the accumulation of EDS1 protein in the plant, so the present inventors further designed experiments to verify whether there is an increase in the cumulative level of EDS1 in the mutant cop1-21 plants.

[0200] The wild-type Col-0, mutant cop1-21 and eds1-2 were cultured under short-day conditions for 3 weeks, and the seedlings were taken for use. At the same time, the wild-type Col-0 and mutant cop1-21 were inoculated with a large amount of Blumeria graminis f. sp. hordei. Five days after inoculation, the seedlings were taken for use.

[0201] The above seedlings were ground into powder in liquid nitrogen, and the total protein was extracted (see Cold Spring Harbor Laboratory Methods, https: / / cshprotocols.cshlp.org / content / 2007 / 1 / pdb.prot4680). After heating and denaturation, the proteins were separated on a 10% SDS-PAGE gel (see the Tetra Cell User Manual of Bio-Rad), and transferred to a membrane (see the Mini Electrophoretic Transfer Cell User Manual of Bio-Rad). Using anti-EDS1 (Agrisera AS132751) as the primary antibody, Western blot analysis was performed. After HRP chemiluminescence color development (XD343079, Thermo), imaging was performed with a Bio-Rad fully automatic chemiluminescence image analysis system. The amount of EDS1 protein was compared according to the concentration of the bands and marked below the bands, and the amount of EDS1 protein in the wild-type Col-0 plants was used as 1. Hybridization with anti-ACTIN antibody was used to show the ACTIN protein to show the consistency of the loading amount during electrophoresis.

[0202] The results of Western blot analysis are shown in Figure 13Compared with the wild-type Col-0 (with the protein amount set to 1), a large accumulation of EDS1 protein was produced in the mutant cop1-21 plants, with the protein amount being 4.58, which was 4.58 times that of the wild-type Col-0. After quantitative inoculation with the powdery mildew G. cichoracearum, the EDS1 protein in both the wild-type Col-0 and the mutant cop1-21 plants increased significantly. Compared with the wild-type Col-0 (with the protein amount of 4.15), the EDS1 protein in the mutant cop1-21 plants increased significantly, with the protein amount being 6.75. Thus, it can be seen that the mutant cop1-21 may cause the accumulation of EDS1 protein by affecting the degradation of EDS1, resulting in the phenotype of resistance to powdery mildew.

[0203] Example 6: The mutant cop1-21 exhibits anti-infection characteristics through the increase of the phytohormone SA

[0204] In order to determine which phytohormones are involved in the powdery mildew resistance of the mutant cop1-21, an experiment on the determination of phytohormone levels in plants was carried out. First, a large number of 3-week-old seedlings of the wild-type Col-0 and the mutant cop1-21 were inoculated with the powdery mildew G. cichoracearum. After 5 days of infection, the leaves were weighed, and 150 mg was taken from each sample. Five replicates were set for each material. After crushing, the phytohormones jasmonic acid (JA), jasmonoyl-isoleucine (JA-Ile), and salicylic acid (SA) were determined. At the same time, the leaf materials of the 3-week-old seedlings of the wild-type Col-0 and the mutant cop1-21 without inoculation with powdery mildew were extracted and the phytohormone determination experiment was carried out simultaneously.

[0205] The determination results of phytohormones are as Figure 14 shown. The content levels of the phytohormones jasmonic acid (JA) and jasmonoyl-isoleucine (JA-Ile) in the Arabidopsis plants of the wild-type Col-0 and the mutant cop1-21 before and after inoculation with the powdery mildew G. cichoracearum did not change significantly, while the content level of salicylic acid (SA) changed significantly. That is, the salicylic acid level of the mutant cop1-21 was significantly higher than that of the wild-type Col-0. After inoculation with powdery mildew, the salicylic acid level of the mutant cop1-21 was also significantly higher than that of the wild-type Col-0. It can be seen that the powdery mildew resistance phenotype of the mutant cop1-21 may be conferred by the increase in the phytohormone salicylic acid.

Claims

1. Use of a COP1 variant protein or its encoding nucleic acid molecule for resisting powdery mildew of Arabidopsis plants, characterized in that: The COP1 variant protein is as shown in SEQ ID NO: 4; the nucleic acid molecule is DNA, cDNA or mRNA encoding the above-mentioned COP1 variant protein.

2. The use according to claim 1, wherein the nucleic acid molecule is as shown in SEQ ID NO: 3.

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