WO2023003176A1 - Gène srfr1 régulateur immunitaire végétal dérivé de la tomate et utilisations de celui-ci - Google Patents

Gène srfr1 régulateur immunitaire végétal dérivé de la tomate et utilisations de celui-ci Download PDF

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WO2023003176A1
WO2023003176A1 PCT/KR2022/008349 KR2022008349W WO2023003176A1 WO 2023003176 A1 WO2023003176 A1 WO 2023003176A1 KR 2022008349 W KR2022008349 W KR 2022008349W WO 2023003176 A1 WO2023003176 A1 WO 2023003176A1
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plant
srfr1
tomato
gene
protein
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김상희
손건희
브엉티우옌
문지윤
김재연
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경상국립대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A01H6/825—Solanum lycopersicum [tomato]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to a tomato-derived plant immunomodulatory factor SRFR1 ( SUPPRESSOR OF rps4-RLD1 ) gene and uses thereof.
  • PTI pattern-triggered immunity
  • ETI effector-triggered immunity
  • PRR Plant receptor (pattern recognition receptor, PRR) protein recognizes the structure and prevents the spread of pathogens in plants through the activation of mitogen-activated protein (MAP) kinase chain reaction, the expression of transcriptional regulators, and the expression of immune-related genes.
  • MAP mitogen-activated protein
  • NLR proteins are divided into the coiled-coil (CC)-NB-LRR (CNL) family and the Toll-interleukin 1-like receptor (TIR)-NB-LRR (TNL) family according to the N-terminal domain.
  • the Arabidopsis SRFR1 ( SUPPRESSOR OF rps4-RLD 1 ) gene was discovered through suppressor screening using wild-type RLD Arabidopsis thaliana, which has a missense mutation in the RPS4 ( Ribosomal protein S4 ) gene.
  • the mutation of SRFR1 in Arabidopsis thaliana Pseudomonas syringae pv .
  • tomato ( Pto ) DC3000 Pseudomonas syringae pv. tomato ( Pto ) DC3000
  • resistance was increased when the corresponding R genes, RPS4 and RPS6 , were mutated, respectively.
  • SRFR1 Since the recessive srfr1 mutant showed similar susceptibility to Pto DC3000 as wild-type RLD, SRFR1 was identified as a negative regulator in ETI. SRFR1 functions as an adapter protein that forms a complex with proteins such as the immune regulator EDS1 (Enhanced disease susceptibility 1) and the TNL-type resistance proteins RPS4, RPS6, and SNC1 (suppressor of npr1-1, constitutive 1). SRFR1 has a TPR (tetratricopeptide repeat) domain similar in nucleotide sequence to Saccharomyces cerevisiae Ssn6 protein, known as a transcriptional repressor regulator.
  • EDS1 Enhanced disease susceptibility 1
  • RPS4, RPS6, and SNC1 uppressor of npr1-1, constitutive .
  • SRFR1 has a TPR (tetratricopeptide repeat) domain similar in nucleotide sequence to Saccharomyces cerevisiae
  • SRFR1 protein interacts with immune chaperone SGT1b (Suppressor of G2 allele of SKP1 homolog B) and TCP family transcription factors. These results support the role of the SRFR1 protein as an adapter protein that negatively regulates the ETI-related transcriptional immune response.
  • the Arabidopsis srfr1 mutant showed a resistance phenotype compared to the wild-type RLD of Arabidopsis thaliana against the insect moth ( Spodoptera exigua ) and the nematode sugar beet cyst nematode ( Heterodera schachtii ).
  • the CRISPR/Cas system consists of an endonuclease Cas protein and a hybrid RNA called single guide RNA (sgRNA).
  • SpCas9 Streptococcus pyogenes Cas9
  • PAM protospacer adjacent motif
  • Cas9 protein RNA-DNA interactions occur, causing Cas9 protein to cause double-strand breaks (DSBs) and error-prone DSB repair while non-homologous end joining (NHEJ) or homology-directed repair (HDR ) through which Indel (Insertion/Deletion) mutation occurs.
  • DSBs double-strand breaks
  • NHEJ non-homologous end joining
  • HDR homology-directed repair
  • Crop genome editing using the CRISPR/Cas system will be an opportunity to accelerate the improvement of crop characteristics against plant pathogens.
  • SRFR1 gene is well conserved as a single copy in many useful crops. Therefore, elucidating the function of SRFR1 in tomato is expected to provide important clues for studying the crop's immune system.
  • Korean Patent No. 101342265 discloses 'disease resistance-related gene CaMLO2 , plant disease resistance search and transformed plants using the same'
  • Korean Patent No. 101007314 discloses 'proteins that regulate plant disease resistance and their gene' is disclosed, but there is no description of the 'tomato-derived plant immunomodulatory factor SRFR1 gene and its use' of the present invention.
  • the present invention was derived from the above needs, and the present inventors prepared a tomato SRFR1 mutant ( slsrfr1 ) using sgRNA and CRISPR / Cas9 targeting two regions of tomato SRFR1 genomic DNA, and the mutant It was confirmed that the expression of a pathogen-related (PR) gene involved in salicylic acid signaling was increased, and it was confirmed that the mutant had increased resistance to Pto DC3000 compared to unmodified tomato plants. However, the necrotrophic fungus Fusarium oxysporum f. sp. It was confirmed that resistance to Lycopersici ( Fusarium oxysporum f. sp. lycopersici ) was reduced compared to unmodified tomato plants. Through this, by confirming that SRFR1 is involved in regulating resistance to parasitic trophic (biotrophic) pathogens and parasitic pathogens in tomato plants, the present invention was completed.
  • PR pathogen-related
  • the present invention provides a method for controlling the disease resistance of a plant, comprising the step of regulating the expression of a gene encoding a protein derived from tomato ( Solanum lycopersicum ) SRFR1 ( SUPPRESSOR OF rps4-RLD1 ) .
  • the present invention comprises the steps of transforming plant cells with a recombinant vector containing a gene encoding tomato-derived SRFR1 protein; and regenerating the transformed plant from the transformed plant cell.
  • the present invention provides a transgenic plant with controlled resistance to plant diseases prepared by the above production method and a transformed seed thereof.
  • the present invention provides a composition for regulating disease resistance of a plant containing, as an active ingredient, a gene encoding tomato-derived SRFR1 protein consisting of the amino acid sequence of SEQ ID NO: 3.
  • SRFR1 which has been studied as a plant immunomodulatory factor in Arabidopsis so far, is also conserved in tomato, and the SRFR1 results of the model plant can be applied to tomato crops and provide grounds for extending to other crops present.
  • SRFR1 is known as a negative regulator in the ETI (effector-triggered immunity) plant immune system mediated by EDS1 by recognizing the effector protein of Pseudomonas, but through the present invention, SRFR1 in both Arabidopsis and tomato is a necrotrophic fungus It was found to be a positive regulator for pathogens. Therefore, by regulating the expression of genes in plants, it is possible to provide new plants with resistance to pathogens, and contribute to the development of the seed industry and the increase in export of food resources.
  • Figure 1a shows the genomic structure and gRNA target location (Target 1, 2) of tomato SRFR1
  • Figure 1b is the target sequence of gRNA
  • Figure 1c is a T-DNA schematic diagram of the pSlSRFR-GE construct
  • Figure 1d is As a result of CAPS analysis of SlSRFR1 sgRNA1- and sgRNA2 -induced G1 mutants, white arrows indicate homozygous mutants.
  • Figure 2 is the result of analyzing the target site in the tomato G0 plant. Vertical dotted lines indicate the cleavage site by SpCas9 for each sgRNA.
  • Figure 5 is a parasitic trophic (biotrophic) pathogen of the G1 generation slsrfr1 plants Pseudomonas syringae pv.
  • tomato DC3000 Pseudomonas syringae pv. tomato DC3000
  • (a) is a photograph showing the disease on the leaves 5 days after inoculation
  • (b) is the result of measuring the number of bacteria. *; P ⁇ 0.01 .
  • the present invention tomato ( Solanum lycopersicum ) Derived SRFR1 ( SUPPRESSOR OF rps4-RLD1 ) A method for regulating disease resistance of a plant, comprising the step of regulating the expression of a gene encoding a protein to provide.
  • the scope of the tomato-derived SRFR1 protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 3 and functional equivalents of the protein.
  • the term "functional equivalent” means at least 70% or more, preferably 80% or more, more preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 3 as a result of addition, substitution or deletion of amino acids. More preferably, it refers to a protein having a sequence homology of 95% or more and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 3.
  • “Substantially homogeneous physiological activity” means an activity that regulates the resistance of plants to plant diseases.
  • the method for controlling plant disease resistance inhibits the expression of the tomato-derived SRFR1 protein-encoding gene to increase resistance to plant diseases caused by biotrophic pathogens, or to necrotrophic pathogens It may be to reduce resistance to plant diseases by, but is not limited thereto.
  • the biotrophic pathogen is preferably Pseudomonas syringae pv. Tomato ( Pto ) DC3000 ( Pseudomonas syringae pv. tomato ( Pto ) DC3000), and the necrotrophic pathogen is preferably Fusarium oxysporum f. sp. It may be Lycopersici ( Fusarium oxysporum f. sp. lycopersici ), but is not limited thereto.
  • VIGS Virus-induced gene silencing
  • RNAi or antisense RNA T-DNA insertion
  • gene correction system or irradiation gene correction system or irradiation
  • Any conventional method in the art that can inhibit the may be possible.
  • VIGS refers to a phenomenon in which, when a plant gene is introduced into a viral vector and then infected, the expression of the endogenous gene of the introduced gene is suppressed. This is a type of PTGS (Post-transcriptional gene silencing), and has the characteristics of post-transcriptional, RNA turnover, and nucleotide sequence-specific.
  • the VIGS vector can be used as a transient expression vector for transient expression in a plant into which a foreign gene has been introduced and a plant expression vector for permanent expression in a plant into which a foreign gene has been introduced.
  • the gene encoding the tomato-derived SRFR1 protein includes both genomic DNA and cDNA.
  • the genomic DNA of tomato-derived SRFR1 of the present invention may include the nucleotide sequence represented by SEQ ID NO: 1
  • the cDNA of tomato-derived SRFR1 may include the nucleotide sequence represented by SEQ ID NO: 2.
  • homologs of the nucleotide sequence represented by SEQ ID NO: 2 are included within the scope of the present invention.
  • the gene is a nucleotide sequence having 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the nucleotide sequence of SEQ ID NO: 2, respectively.
  • the "percentage of sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. may include additions or deletions (i.e., gaps) compared to (not including).
  • the present invention also relates to the present invention.
  • Regenerating the transformed plant from the transformed plant cell provides a method for producing a transgenic plant with controlled resistance to plant diseases, including.
  • the term "recombinant” refers to a cell that replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a protein encoded by a heterologous peptide, or a heterologous nucleic acid.
  • Recombinant cells can express genes or gene segments not found in the cell's native form, either in sense or antisense form.
  • a recombinant cell may also express a gene found in the cell in its natural state, but the gene has been reintroduced into the cell by artificial means as a modified one.
  • vector is used to refer to DNA fragment(s), nucleic acid molecules, which are delivered into cells. Vectors replicate DNA and can reproduce independently in host cells.
  • delivery vehicle is often used interchangeably with “vector”.
  • expression vector refers to a recombinant DNA molecule comprising a coding sequence of interest and appropriate nucleic acid sequences necessary to express the operably linked coding sequence in a particular host organism.
  • the vectors of the present invention may typically be constructed as vectors for cloning or expression.
  • the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.
  • a strong promoter capable of promoting transcription eg, pL ⁇ promoter, Trp promoter, Lac promoter, T7 promoter, Tac promoter, etc.
  • It typically includes a ribosome binding site for initiation of translation and a transcription/translation termination sequence.
  • Escherichia coli is used as the host cell, the E. coli tryptophan biosynthesis pathway promoter and operator regions and the leftward promoter of phage ⁇ (pL ⁇ promoter) can be used as control regions.
  • the promoter may be promoters suitable for transformation, preferably CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter, preferably CaMV 35S promoter. However, it is not limited thereto.
  • promoter refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in a plant cell.
  • a “constitutive promoter” is a promoter that is active under most environmental conditions and states of development or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of transformants may be made by various tissues at various stages. Thus, constitutive promoters do not limit selection possibilities.
  • the recombinant vector of the present invention can be constructed by methods well known to those skilled in the art.
  • the method includes in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like.
  • the DNA sequence can be effectively linked to a suitable promoter in an expression vector to direct mRNA synthesis.
  • the vector may include a ribosome binding site and a transcription terminator as a translation initiation site.
  • a preferred example of a plant expression vector is the Ti-plasmid vector, which when present in a suitable host, such as Agrobacterium tumefaciens , is capable of transferring a part of itself, the so-called T-region, into plant cells.
  • a suitable host such as Agrobacterium tumefaciens
  • Another type of Ti-plasmid vector (see EP 0 116 718 B1) is currently used to transfer hybrid DNA sequences into plant cells, or protoplasts, from which new plants can be produced that properly integrate the hybrid DNA into the plant's genome.
  • a particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838.
  • viral vectors such as those that can be derived from double-stranded plant viruses (eg, CaMV) and single-stranded viruses, gemini viruses, and the like.
  • CaMV double-stranded plant viruses
  • it may be selected from incomplete plant viral vectors. The use of such vectors can be particularly advantageous when properly transforming a plant host is difficult.
  • a preferred example of the recombinant vector of the present invention may be a vector comprising a gRNA sequence targeting a gene encoding tomato-derived SRFR1 protein and a sequence encoding a Cas9 endonuclease.
  • Recombinant expression vectors may preferably contain one or more selectable markers.
  • the marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing transformed cells from non-transformed cells.
  • the marker gene may be a dominant drug resistance gene, but is not limited thereto.
  • Any host cell known in the art can be used as the host cell capable of stably and continuously cloning and expressing the vector of the present invention.
  • E. coli JM109 E. coli BL21
  • Bacillus genus strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110
  • Bacillus subtilis Bacillus thuringiensis
  • enterobacteriaceae and strains such as Salmonella typhimurium , Serratia marcescens and various Pseudomonas sp.
  • yeast eg, Saccharomyce cerevisiae
  • insect cells eg, human cells
  • human cells eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7 , 293, HepG2, 3T3, RIN and MDCK cell lines
  • plant cells preferably plant cells.
  • Plant transformation refers to any method of transferring DNA into a plant. Such transformation methods need not necessarily have a period of regeneration and/or tissue culture. Transformation of plant species is now common for plant species including both dicotyledonous as well as monocotyledonous plants.
  • any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. Methods include the calcium/polyethylene glycol method for protoplasts (Negrutiu I. et al., 1987, Plant Mol. Biol. 8, 363-373), electroporation of protoplasts, microinjection into plant elements (Crossway A. et al. Gen. Genet. In Paciens mediated gene transfer, it can be suitably selected from infections by (incomplete) viruses (EP 0 301 316) and the like. Preferred methods according to the present invention include Agrobacterium mediated DNA delivery.
  • any method known in the art may be used for regenerating the transgenic plant from the transformed plant cell.
  • Transformed plant cells must regenerate into whole plants.
  • Techniques for regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species.
  • a “plant cell” used for plant transformation can be any plant cell.
  • a plant cell is a cultured cell, cultured tissue, cultured organ or whole plant.
  • Plant tissue refers to differentiated or undifferentiated plant tissues, such as but not limited to roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used in culture, i.e., single cells, protoplasts. (protoplast), shoot and callus tissue.
  • the plant tissue may be in planta or may be in organ culture, tissue culture or cell culture.
  • Parasitic trophic pathogens and parasitic pathogens are as described above.
  • the present invention also provides transgenic plants and their transformed seeds having controlled resistance to plant diseases prepared by the above production method.
  • Transgenic plants with controlled resistance to plant diseases inhibit the expression of the SRFR1 protein-coding gene to increase resistance to plant diseases caused by parasitic trophic pathogens or to increase resistance to plant diseases caused by parasitic pathogens It is characterized by this reduction.
  • the plants are tomato, Arabidopsis, potato, eggplant, tobacco, red pepper, burdock, crown daisy, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, water parsley, parsley, Chinese cabbage, cabbage, mustard, watermelon, melon, cucumber, It may be a dicotyledonous plant such as pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, or pea, or a monocotyledonous plant such as rice, barley, wheat, rye, corn, sugar cane, oats, and onion, preferably a dicotyledonous plant. It may be, more preferably may be a tomato plant, but is not limited thereto.
  • the present invention provides a composition for regulating disease resistance of a plant containing, as an active ingredient, a gene encoding tomato-derived SRFR1 ( SUPPRESSOR OF rps4-RLD1 ) protein consisting of the amino acid sequence of SEQ ID NO: 3.
  • the composition of the present invention contains, as an active ingredient, a tomato-derived SRFR1 protein coding gene capable of controlling the resistance of tomato plants to plant diseases, and inhibiting the expression of the gene can control the disease resistance of the plants.
  • the target site of the SpCas9-gRNA complex was investigated at the N-terminal region of SlSRFR1 using the CRIPSR -P v2.0 program (http://crispr.hzau.edu.cn/CRISPR2/).
  • two gRNAs were selected considering the on-score, which is a numerical value that can cause genome editing, the secondary structure of gRNA, the GC nucleotide content of the target site, and the off-target sequence.
  • the two gRNAs were linked with the Arabidopsis U6 promoter (originated from Addgene #46968), scafford RNA and poly T through primer dimerization, and the entire gRNA was Level 1 plasmid through the Golden Gate cloning system.
  • Each gRNA module created was cloned into pAGM4723 using the Golden Gate cloning system along with a plant selectable marker (Addgene #51144), SpCas9 (Addgene #49771), and a linker (Addgene #48019) to prepare a construct.
  • the site was named pSlSRFR1-GE.
  • Tomato transformation and plant selection were performed with reference to previous studies (Plant Cell Rep. 2021 Jun; 40(6):999-1011).
  • Tomato cultivar M82 Solanum lycopersicum cv. M82
  • the pSlSRFR1-GE plasmid was transformed into Agrobacterium tumefaciens GV3101 (MP90) by electroporation.
  • Agrobacterium having pSlSRF1-GE cultured at 28 ° C for 18 hours was transferred to 30 ml of LB medium (including 50 mg L -1 kanamycin and 10 mg L -1 gentamicin), and the OD 600 value was about 0.8 to 1 cultured until The Agrobacterium was centrifuged at 3,000 rpm for 15 minutes and the pellet was washed in 30 ml of ABM-MS [1.106 g K 2 HPO 4 , 0.565 g KH 2 ] containing 200 ⁇ M acetosyringone (#D134406, Sigma, USA).
  • Tomatoes were transformed by incubating the suspension for one hour at 28° C. and co-incubating tomato cotyledon explants with Agrobacterium for 20 minutes. Tomato cotyledon explants were transferred to ABM-MS containing 200 ⁇ M acetosyringone and cultured for 2 days at 25° C. in the dark.
  • Agrobacterium attached to the explants was washed by stirring in 500 mg ⁇ L -1 titentin solution for 2 minutes. The water remaining on the explants was completely removed using Whatman paper, and the selective medium [SEL4-70; 4.4g MS salts + B5, 30g maltose, 0.05mg IAA, 0.976g MES, 0.5mg zeatin ribose trans-isomer, 70mg kanamycin, 300mg timentin, 1mM putrescine (pH 5.7), 8g agar for 1L] to induce callus Callus was transferred to a new selection medium (SEL4-70) every 2 weeks until complete stems appeared.
  • the explants from which the stems emerged are rooted induction medium [RIM; 2.2g MS salts + B5, 20g sucrose, 0.1mg NAA, 0.3mg IBA, 300mg Timentin (pH 5.7), 8g agar for 1L] and cultured until roots were induced. Healthy plants are grown at 25°C, 16 hours light/8 hours dark conditions.
  • Genomic DNA isolation was performed with reference to Pater et al. (Plant Biotechnol J. 2009 Oct;7(8):821-835). Using a cork borer No. 5, two leaf pieces from G0 and G1 generation tomato plants were put into a tube, frozen in liquid nitrogen, and ground using beads in a mixer mill (#MM301, Retsch, Germany) for 1 minute. . 300 ⁇ l of 2X CTAB extraction buffer (0.1M Tris, 2% CTAB, 1.4M NaCl, and 20mM EDTA) was added to the powder and reacted at 65° C. for 20 minutes. 300 ⁇ l of chloroform was additionally added to the tube, mixed vigorously, and centrifuged at 14,000 rpm for 5 minutes.
  • 2X CTAB extraction buffer 0.1M Tris, 2% CTAB, 1.4M NaCl, and 20mM EDTA
  • the SlSRFR1 target region was amplified by PCR in G0 or G1 generation tomatoes and sequencing was performed.
  • the above results were confirmed by ICE (Inference of CRISPR Edits, https://ice.synthego.com/#/) analysis to see how many Indel mutations occurred in G0 or G1 generation tomatoes.
  • ICE Inference of CRISPR Edits, https://ice.synthego.com/#/
  • Miseq sequencing was performed and analyzed with the Cas-Analyzer program (http://www.rgenome.net/).
  • a 945 bp PCR fragment was amplified using the SlSRFR1-F1/SlSRFR1-R2 primer set, and primers (MiSeq-1-F2/MiSeq-1-R2 and MiSeq-1-R2 and MiSeq-2-F2/MiSeq-2-R2) were used to amplify DNA fragments of 155bp and 150bp in size through secondary PCR.
  • the tertiary PCR fragment was amplified using dual index adapter (a combination of D501-D508 and D701-D712) primers provided by the MiSeq sequencing service (MiniSeqTM System, Illumina, USA), and the degree of indel mutation was analyzed by Miseq sequencing.
  • Pseudomonas cold dog pv. Pathogenesis analysis by tomato DC3000 was performed according to a previous study (Scalschi, L. et al ., PLoS One 2014, 9:e106429).
  • Pto DC3000 containing the pVSP61 empty vector grown in Pseudomonas medium was diluted to 2x10 8 CFU/ml, and the third or fourth leaf of a 6-week-old tomato plant was treated with the diluted solution for 30 seconds and maintained at high humidity. To do this, it was wrapped in a plastic bag and maintained for 5 days.
  • Fusarium oxysporum f. sp. Pathogenesis analysis by Fusarium oxysporum f. sp. lycopersici (hereinafter referred to as FOL) follows previous studies (Kostov, K. et al ., Biotechnol. Biotechnol. Equip. 2009, 23(1):1121-1125). performed according to After culturing FOL in potato dextrose (#254920, BD Difco, USA) in the dark at 30 ° C, it was grown for 5 days at 25 ° C for 16 hours light / 8 hours in the dark and used for the experiment. Using Corkborder No. 1, a fungus grown in the medium was made into a plug and placed on 6-week-old tomato leaves, and left at 25 ° C. for 3-8 days under 16-hour light / 8-hour dark conditions.
  • FOL Fusarium oxysporum f. sp. Pathogenesis analysis by Fusarium oxysporum f.
  • the dyed leaves were soaked in 99% ethanol for one day to remove the pigment.
  • the degree of development of fungal hyphae was visualized using a fluorescence microscope.
  • Protein immunoblot analysis was performed using ⁇ -PR1 antibody (1:10,000 dilution, #AS10 687, Agrisera, SWEDEN) or ⁇ -Actin antibody (1:10,000 dilution, #AS13 2640, Agrisera), and Clarity Western ECL Substrate (#1705061, Bio-Rad, USA) and SuperSignalTM West Femto Maximum Sensitivity Substrate (#34094, Thermo Scientific, USA).
  • SRFR1 is a single-copy gene in Arabidopsis thaliana, and Arabidopsis SRFR1 is mainly composed of 11 TPR (tetratricopeptide repeat) domains involved in protein-protein interactions.
  • the Blast algorithm was used to annotate the tomato proteome, and Solyc02g09280 (SlSRFR1) with 65% similarity to Arabidopsis SRFR1 was identified.
  • SlSRFR1 encodes a protein consisting of 1,055 amino acids, with two TPR domains located at the N-terminus and nine TPR domains located at the center.
  • sgRNA was designed based on standard conditions, and the 5' end of the open reading frame (ORF) of SlSRFR1 was selected as a site for correction (Table 2).
  • gRNA target site for SlSRFR1 correction Name gRNAs Target position on genome Number of off-targets ( ⁇ 4MMs) sgRNA1 GTAACTTTCGACGCCATCG (SEQ ID NO: 4) 5'UTR and CDS One sgRNA2 ATTGACTATAGCAAAACGCT (SEQ ID NO: 5) CDS 3
  • a 1 bp or 11 bp deletion of the TPR domain resulted in a premature stop codon.
  • mutations in slsrfr1-2 and slsrfr1-4 were identified in the initiation codon.
  • Sanger sequencing analysis of the purified PCR amplicons revealed that off-target sites were not identified in all subjects.

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Abstract

La présente invention concerne un procédé de régulation de la résistance aux maladies de plantes en régulant l'expression d'un gène codant pour une protéine de suppression de rps4-RLD1 (SRFR1) dérivée de la tomate (Solanum lycopersicum) constituée d'une séquence d'aminoacides avec un SEQ ID NO : 3.
PCT/KR2022/008349 2021-07-21 2022-06-14 Gène srfr1 régulateur immunitaire végétal dérivé de la tomate et utilisations de celui-ci WO2023003176A1 (fr)

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KR20180000282A (ko) * 2016-06-22 2018-01-02 서울대학교산학협력단 토마토 유래 sra1 유전자를 이용한 식물의 해충 저항성을 증대시키는 방법 및 그에 따른 식물체

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Publication number Priority date Publication date Assignee Title
KR20180000282A (ko) * 2016-06-22 2018-01-02 서울대학교산학협력단 토마토 유래 sra1 유전자를 이용한 식물의 해충 저항성을 증대시키는 방법 및 그에 따른 식물체

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Understanding the mechanisms of plant immune response and application thereof in crop resistance engineering", FINAL REPORT OF NEXT GENERATION BIO GREEN 21, SYSTEMS & SYNTHETIC AGROBIOTECH PROGRAM; INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY; PJ013449, vol. 12, no. 31, 1 February 2021 (2021-02-01), XP093027183 *
DATABASE PROTEIN ANONYMOUS : "suppressor of RPS4-RLD 1 [Solanum lycopersicum]", XP093027182, retrieved from NCBI *
KWON SOON IL, SANG HEE KIM, SAIKAT BHATTACHARJEE, JAE-JONG NOH, WALTER GASSMANN: "SRFR1, a suppressor of effector‐triggered immunity, encodes a conserved tetratricopeptide repeat protein with similarity to transcriptional repressors", THE PLANT JOURNAL, vol. 57, pages 109 - 119, XP093027180, DOI: 10.1111/j.1365-313X.2008.03669.x *
LI YINGZHONG, LI SHUXIN, BI DONGLING, CHENG YU TI, LI XIN, ZHANG YUELIN: "SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity", PLOS PATHOGENS, vol. 6, no. 9, 16 September 2010 (2010-09-16), pages e1001111, XP093027178, DOI: 10.1371/journal.ppat.1001111 *
SON GEON HUI, MOON JIYUN, SHELAKE RAHUL MAHADEV, VUONG UYEN THI, INGLE ROBERT A., GASSMANN WALTER, KIM JAE-YEAN, KIM SANG HEE: "Conserved Opposite Functions in Plant Resistance to Biotrophic and Necrotrophic Pathogens of the Immune Regulator SRFR1", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 22, no. 12, 1 January 2021 (2021-01-01), pages 6427, XP093027176, DOI: 10.3390/ijms22126427 *

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