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WO2021124323A1 - Moyen et procédés de fourniture d'une résistance contre les mauvaises herbes parasitaires - Google Patents

Moyen et procédés de fourniture d'une résistance contre les mauvaises herbes parasitaires Download PDF

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WO2021124323A1
WO2021124323A1 PCT/IL2020/051289 IL2020051289W WO2021124323A1 WO 2021124323 A1 WO2021124323 A1 WO 2021124323A1 IL 2020051289 W IL2020051289 W IL 2020051289W WO 2021124323 A1 WO2021124323 A1 WO 2021124323A1
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cas9
tomato
parasitic
plants
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Radi Aly
Vinay KUMAR BARI
Jacline ABU-NASSAR
Amit Gal-On
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Israel Ministry of Agriculture and Rural Development
Agricultural Research Organization of Israel Ministry of Agriculture
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Agricultural Research Organization of Israel Ministry of Agriculture
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • 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
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    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

  • the present invention relates to the field of agriculture, and more particularly the present invention concerns parasitic weed control and a method for providing resistance to tomato plants against the parasitic weed P. aegyptiaca upon mutation of the strigolactone-biosynthesis gene CCD8 using the CRISPR/Cas9 technique.
  • the life cycle of P. aegyptiaca is divided into two stages, pre-parasitic and parasitic.
  • the pre- parasitic stage consists of seed preconditioning followed by germination.
  • the germination of parasite seeds is triggered by a highly specialized detection system for strigolactones which are exuded by the host’s roots.
  • the parasitic stage initiates with the parasite developing a special intrusive organ, i.e. the haustorium, that connects directly to the vascular system of the host.
  • the broomrape seedling grows into a structure known as tubercle and after 4-5 weeks of tubercle growth, a floral meristem is produced, which emerges above the ground to produce flowers and seeds.
  • Strigolactones are plant hormone required for shoot branching and used as signaling molecules for the rhizosphere microflora. Strigolactones are produced in all green lineages of the plant kingdom and their synthesis starts with the all trans b-carotene, a carotenoid molecule which produces 9-cis ⁇ -carotene by the activity of D27, after that carotenoid cleavage dioxygenases 7 ( CCDT) converts it into 9 cis b-apo 10’- carotenal and finally carotenoid cleavage dioxygenases 8 ( CCD8 ), leading to the production of carlactone, which is then converted by cytochrome P450 enzymes ⁇ MAXI) into various strigolactones .
  • CCDT carotenoid cleavage dioxygenases 7
  • CCD8 carotenoid cleavage dioxygenases
  • strigolactone acts as a host-detection cue for symbiotic arbuscular mycorrhizal fungi and stimulates seed germination of parasitic plants.
  • strigolactone such as strigol, 5-deoxystrigol, sorgolactone, solonacol, dideoxyorobanchol, orobanchol and others, are known as germination stimulants for root parasites.
  • Host resistance to the Orobanchaceae root parasite Striga has been observed in crops with altered strigolactone production.
  • the altered strigolactone production conferred resistance in the host by reducing the germination of parasite seeds.
  • previous studies found that the tomato SL-ORT1 mutagenized by fast-neutron displays a high resistance to Phelipanche and Orobanche spp, which results from its inability to produce and secrete strigolactones regarded as natural germination stimulants to the rhizosphere.
  • CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9
  • CRISPR/Cas9 permits the direct introduction of mutations conferring resistance in crop plants, without traditional backcrosses or plant breeding.
  • Cas9-DNA scissors makes site-specific double-strand cut in the genome, while Cas9 base editor have ability to alter a specific nucleotide into another, inducing modifications at targeted locus through homologous recombination or non-homologous end-joining repair mechanisms.
  • the most frequently used CRISPR/Cas9 system, type II has three components: Cas9 endonuclease, CRISPR RNA (crRNA) and trans activating crRNA (tracrRNA).
  • Cas9-mediated DNA cleavage is guided by a tracrRNA:crRNA duplex that is complementary to the crRNA.
  • the tracrRNA: crRNA complex is fused into a chimeric single guide RNA (sgRNA) containing an 18 to 20-nucleotide (nt) sequence which determines the target DNA sequence.
  • sgRNA chimeric single guide RNA
  • nt 18 to 20-nucleotide sequence which determines the target DNA sequence.
  • the NGG protospacer adjacent motif (PAM) that is present at 3 '-end of the target sequence is recognized by the CRISPR/Cas9 system.
  • CRISPR/Cas9 Use of CRISPR/Cas9 has been reported as the most effective tool for nucleotide sequence modification or editing in numerous crop species, including tomato, rice, cotton, maize, soybean, and tobacco.
  • Mahfouz, M. et al managed to engineer the rice plant’s architecture through genomic editing of OsCCD7 gene using the CRISPR/Cas9 technique, and thus, generated plants with decreased strigolactone content and reduced Striga hermonthica germination (See Butt, H., Jamil, M., Wang, J. Y., Al-Babili, S., & Mahfouz, M. “Engineering plant architecture via CRISPR/Cas9-mediated alteration of strigolactone biosynthesis”. BMC plant biology, 18(1), 174, 2018).
  • Hershenhorn, J. et al showed that the roots of the fast-neutron-mutagenized tomato mutant SL-ORT1, which is resistant to broomrape, lacks strigolactones orobanchol, solanacol, and didehydro-orobanchol isomer.
  • the researchers did not use molecular tools to generate resistant plants (See “Strigolactone Deficiency Confers Resistance in Tomato Line SL-ORT1 to the Parasitic Weeds Phelipanche and Orobanche spp”. Phytopathology 101, 213-222, 2011).
  • Fig.l depicting a schematic presentation of the binary plant expression construct used for Cas9 and CCD8sgRNA expression
  • Fig.2 depicting a restriction analysis and sequence alignment of CCD8 Cas9-edited tomato lines of the present invention
  • Fig.3 depicting a restriction analysis and sequence alignment of CCD8 Cas9-edited tomato lines of the present invention
  • Fig.4 graphically depicting different comparative traits of CCD8 Cas9-edited tomato lines of the present invention
  • Fig.5 graphically depicting different comparative traits of CCD8 Cas9-edited tomato lines of the present invention
  • Fig.6 depicting a graphical presentation of the weed resistance trait of the CCD8 Cas9-edited tomato lines of the present invention
  • Fig.7 graphically depicting the relative orobanchol content of CCD8 Cas9 -edited tomato lines of the present invention
  • Fig.8 graphically depicting sequences of the CCD8 Cas9 mediated mutated T1 plants of the present invention
  • Fig.9 depicting the carotenoid contents in CCD8 Cas9 mutants and relative transcript expression of three candidate genes in CCD8 Cas9 mutated lines of the present invention
  • Fig.10 depicting a schematic presentation of the mutation presence in TO-generation of ccos Cas9 edited lines of the present invention
  • Fig.ll depicting a schematic presentation of identification of transgene (Cas9) free plants in T1 generation using different CCDS Cas9 mutants of the present invention
  • Fig.12 depicting a schematic presentation of sequence chromatogram of potential off-target of the CCDS sgRNA.
  • Fig.13 depicting a graphical presentation of phenotypical characterization of ccos Cas9 mutants of the present invention.
  • the present invention provides a transgenic tomato plant with increased resistance to the parasitic weed P. aegyptiaca.
  • the present invention provides a method of generating resistant tomato plants by disrupting the strigolactone -biosynthesis gene CCD8 using the genome-editing CRISPR/Cas9 technique.
  • the parasitic weeds are selected from a group consisting of: the Balanophoraceae family, the Orobanchaceae family, the Rafflesiaceae family, the Loranthaceae family, the Hydnoroideae family, the Mystropetalaceae family, the Santalaceae family, the Lauraceae family, the Convolvulaceae family, the Viscaceae family, the Misodendraceae family, the Olacaceae family, the Opiliaceae family, the Schoepfiaceae family and any combination thereof.
  • the parasitic weeds are selected from a group consisting of: the Balanophoraceae family, the Orobanchaceae family, the Rafflesiaceae family, the Loranthaceae family, the Hydnoroideae family, the Mystropetalaceae family, the Santalaceae family, the Lauraceae family, the Convolvulaceae family, the Viscaceae family, the Misodendraceae
  • the parasitic weeds are selected from a group consisting of: the Balanophoraceae family, the Orobanchaceae family, the Rafflesiaceae family, the Loranthaceae family, the Hydnoroideae family, the Mystropetalaceae family, the Santalaceae family, the Lauraceae family, the Convolvulaceae family, the Viscaceae family, the Misodendraceae family, the Olacaceae family, the Opiliaceae family, the Schoepfiaceae family and any combination thereof.
  • the single guide RNA sequence comprises the nucleic acid sequence TTCATTCAGCTCATCCAG.
  • the transforming step is selected from a group consisting of: the Agrobacterium- mediated transformation method, particle bombardment, injection, viral transformation, in planta transformation, electroporation, lipofection, sonication, silicon carbide fiber mediated gene transfer, laser microbeam (UV) induced gene-transfer, co-cultivation with the explants tissue and any combination thereof.
  • testing is selected from a group consisting of: PCR amplifications, restriction assays, Sanger sequencing, pyrosequencing, sequencing by synthesis (Ilumina), Ion Torrent sequencing and any combination thereof.
  • It is another object of the present invention to disclose a method for verifying a resistance to parasitic weeds conferred to ccos Cas9 -edited tomato plants comprising steps of:
  • the parasitic weeds are selected from a group consisting of: the Balanophoraceae family, the Orobanchaceae family, the Rafflesiaceae family, the Loranthaceae family, the Hydnoroideae family, the Mystropetalaceae family, the Santalaceae family, the Lauraceae family, the Convolvulaceae family, the Viscaceae family, the Misodendraceae family, the Olacaceae family, the Opiliaceae family, the Schoepfiaceae family and any combination thereof. It is another object of the present invention to disclose the method as defined in any one of the above, wherein the parasitic weed is Phelipanche aegyptiaca.
  • the term “about” refers to any value being up to 25% lower or greater the defined measure.
  • the term “parasitic weeds” refers to any plant which derives some or all of its nutritional requirement from another living plant.
  • CRISPR/Cas9 refers to a genome-editing technique, wherein a synthetic guide RNA sequence complexed with the CAS9 nuclease is delivered to a gene of interest, aiming to specifically modify its sequence.
  • CCD8 Cas9-edited tomato plants refers to any tomato plant, whose genome was edited via the CRISPR/CAS9 technique. More specifically, a single guide RNA sequence targets the carotenoid cleavage dioxygenases 8 (CCD8) gene of the tomato plants, and disrupts its translation.
  • CCD8 Cas9-edited tomato plants refers to any tomato plant, whose genome was edited via the CRISPR/CAS9 technique. More specifically, a single guide RNA sequence targets the carotenoid cleavage dioxygenases 8 (CCD8) gene of the tomato plants, and disrupts its translation.
  • CCD8 Cas9-edited tomato plants refers to any tomato plant, whose genome was edited via the CRISPR/CAS9 technique. More specifically, a single guide RNA sequence targets the carotenoid cleavage dioxygenases 8 (CCD8) gene of the tomato plants, and disrupts its translation.
  • CCD8 Cas9-edited tomato plants refers to any tomato plant,
  • CCB8 sgRNA refers to any guide RNA sequence configured to target the tomato carotenoid cleavage dioxygenases 8 (CCD8) gene.
  • the CCDS sgRNA of the present invention is a component in a CRISPR/CAS9 system, specifically designed to disrupt the activity of the carotenoid cleavage dioxygenases 8 (CCD8) gene, and create plants with enhanced resistance to parasitic weeds.
  • CCB8 Cas9 knockout phenotype refers to any phenotype exhibited by the CCD8 Cas9-edited tomato plants. This phenotype may manifest as dwarfing, excessive shoot branching, adventitious root formation, increase in lateral roots, reduced fruit sizes, reduced orobanchol content, increase in total carotenoids level or any combination thereof.
  • PAM protospacer adjacent motif
  • rootstock refers to part of a plant comprising the stem and/or underground part or rooting system of that plant and onto which a scion, cutting or bud is intended to be grafted.
  • progeny refers in a non-limiting manner to offspring or descendant plants of the genome-edited tomato plants of the present invention.
  • tomato plant refers to a plant of the genus Solanum, preferably to plants of the species Solanum lycopersicum.
  • locus refers to a specific place or places or region or a site on a chromosome where for example a gene or genetic marker element or factor is found. In specific embodiments, such a genetic element is contributing to a trait.
  • trait refers to a characteristic or phenotype.
  • a phenotypic trait may refer to the appearance or other detectable characteristic of an individual, resulting from the interaction of its genome, proteome and/or metabolome with the environment.
  • a branching shoot trait relates a disruption in the CCD8 gene.
  • a trait may be inherited in a dominant or recessive manner, or in a partial or incomplete-dominant manner.
  • a trait may be monogenic (i.e. determined by a single locus) or polygenic (i.e. determined by more than one locus) or may also result from the interaction of one or more genes with the environment.
  • a dominant trait results in a complete phenotypic manifestation at heterozygous or homozygous state; conventionally, a recessive trait manifests itself only when present at homozygous state.
  • the phrase "resistance” refers to the ability of a plant to restrict the growth of a parasitic weed growing next to it, or to develop normally unaffected and uninterrupted by the presence of the parasitic weed.
  • transgenic tomato plants Solanum lycopersicum L.
  • CCD8 Solyc08g066650
  • This method comprises the following steps: (a) designing a CCDS sgRNA construct using the chimeric single guide RNA (sgRNA) cassette in the pENTR vector; (b) recombining the pENTR vector into the pDest vector to target the second exon of CCD8 (position 716- 733bp in the coding region) with a Bsrl restriction site located next to the protospacer adjacent motif (PAM); (c) transforming the tomato plants using the Agrobacterium-mediated transformation method; (d) genetically assessing the mutations caused by the genome editing in the different lines showing different kinds of genome editing events in the TO generation for the CCDS Cas9 locus; (e) growing TO transgenic tomato lines to maturity; (f) allowing self-pollination of the TO tomato lines to generate T1 progeny and to avoid somatic mutations; (g) genetically testing the sgRNA construct using the chimeric single guide RNA (sgRNA) cassette in the pENTR vector; (b) recombining
  • This method comprises the following steps: (a) triggering independent transgenic tomato plants from T1 lines representing the ccos Cas9 knockout phenotypes with P. aegyptiaca seeds; (b) randomly selecting T1 progeny of each line, irrespective of their zygosity (homozygous or biallele); (c) transplanting the selected T1 tomato plants in small pots containing soil infested with P.
  • aegyptiaca seeds (20 mg/kg soil); (d) growing the selected T1 tomato plants for about 3 months in a greenhouse; (e) measuring the resistance of the CCDS Cas9 mutated lines by counting only fresh and viable parasite tubercles which are larger than 2 mm in diameter from each plant.
  • the strigolactone -biosynthesis gene CCD8 (Solyc08g066650) was disrupted in tomato strigolactones are synthesized from plant carotenoids via a pathway involving CCD7 and CCD8.
  • the CCD8 sgRNA construct was designed using the single sgRNA cassette in the pENTR vector, which was then recombined into the pDest vector to target the second exon of CCD8 (position 716-733bp in the coding region) with a Bsrl restriction site located next to the protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • Fig. 1 depicting a schematic presentation of the binary plant expression construct used for Cas9 and CCD8 sgRNA expression.
  • the strong constitutive 2X35SQ promoter (CaMV- 2x35S promoter with the omega enhancer sequence) was used to express Arabidopsis codon-optimized Cas9 and the Arabidopsis U6-26 promoter was used to express CCDS sgRNA.
  • Fig. IB a schematic representation of the tomato SICCD8 genomic map and location of the CCDS sgRNA target site are shown.
  • the target sequence of CCDS sgRNA is depicted, the PAM is marked at the right end of the sequence (dashed grey line), the Bsrl restriction site and the sequence are marked by a black triangle and underlined, black arrows indicate the primer positions (S1CCD8-Int-F and S1CCD8-Int-R) used for PCR amplification, and the white arrow indicates location of CCDS sgRNA target site.
  • the loss of the Bsrl restriction enzyme site that might arise due to imprecise non-homologous end-joining repair was evaluated.
  • the CCDS Cas9 target region was PCR-amplified using genomic DNA from the TO lines (individual transformants) and then digested with Bsrl (a site that would be disrupted if Cas9-mediated genome editing occurred in this location) and products on a 2% agarose gel were assessed.
  • FIG. 2-Fig.3 depicting a restriction analysis and sequence alignment of CCD8 Cas9- edited tomato lines of the present invention.
  • Fig. 2A a restriction digestion assay using PCR fragments of CCDS Cas9 targeted region in TO lines is presented. The amplified PCR fragments from genomic DNA of independent transgenic plants were subjected to /i.vrl-restriction digestion. Lines 1, 2, 5 and 11 represent independent TO transgenic plants; + : Bsrl added; without Bsrl.
  • Fig. 2B PCR product sequence alignments of the selected TO lines with the wild-type genome sequences (WT) are depicted.
  • WT wild-type genome sequences
  • PAM protospacer adjacent motif
  • Bsrl site is shown in bold underlined, black triangle indicates the digestion site for Bsrl;
  • DNA deletions are presented by black dots and deletion sizes (nt) are marked on the right side of the sequences.
  • ccos Cas9 target regions from TO lines 5 and 11 were amplified by PCR and cloned into the TA cloning vector. Sanger sequencing of positive clones aligned with wild- type sequence, and type of mutation (indels) detected are presented on the right side of the sequences. Nucleotide sequence inside the red box encodes for stop codon. Reference is now made to Fig.
  • FIG. 3A where a restriction digestion assay using PCR fragments of CCDS Cas9 targeted region in T1 generation is depicted.
  • the amplified PCR fragment from genomic DNA of independent transgenic plants was subjected to Bsrl restriction digestion; +, Bsrl added; -, without Bsrl.
  • Fig. 3B PCR product sequence alignments of the selected T1 lines is depicted.
  • PAM is shown in red; Bsrl site is shown in bold underlined, black triangle indicates digestion site for Bsrl; DNA deletions are shown in black dots and deletion sizes (nt) are marked on the right side of the sequences; Nucleotide sequence inside the red box encodes for stop codon.
  • strigolactones regulate plant growth and morphological architecture Strigolactone-deficient mutants are known to exhibit an increase in shoot branching, lateral roots and overall dwarfing.
  • CCDS Cas9 mutated lines A similar phenotypic profile in CCDS Cas9 mutated lines was observed, such as highly branched shoots, increased lateral roots, decreased shoot heights and reduced fruit sizes as compared to the wild type plants.
  • Figs. 4-5 graphically illustrating differences between The ccos Cas9 edited tomato plants and WT plants.
  • Fig. 4A depicts a quantitative estimate of secondary branches in one month old ccos Cas9 mutated tomato plants (lines 1,2,5 and 11) compared to a WT plant, wherein in all edited lines the number of secondary branches is higher in comparison with WT ⁇ error bars indicate +SD n(7) ⁇ .
  • Fig. 4B depicts plants’ height of the CCDS Cas9 mutated tomato plants (lines 1,2,5 and 11) compared to a WT plant after 3 month of growth ⁇ error bars indicate +SD, n(ll) ⁇ . The results indicated that the edited tomato lines are shorter than WT plants.
  • Fig. 4A depicts a quantitative estimate of secondary branches in one month old ccos Cas9 mutated tomato plants (lines 1,2,5 and 11) compared to a WT plant, wherein in all edited lines the number of secondary branches is higher in comparison with WT ⁇
  • FIG. 5A depicts the size of mature fruits of CCDS Cas9 mutated tomato plants (lines 1,2,5 and 11) compared to a WT plant ⁇ error bars indicate +SD n(22) ⁇ .
  • Fig. 5B graphically depicts the number of fruit produced by ccos Cas9 mutated lines compared to WT tomato plant ⁇ error bars indicate +SD n(ll) ⁇ .
  • the different small letters not connected by same letter on each bar indicate statistically significant differences compared to wild-type plants (p-value ⁇ 0.05; Student’s t-test using JPM-14 software).
  • Fig. 6 a graphical presentation of the resistance trait of CCD8 Cas9 edited tomato lines of the present invention are depicted, respectively.
  • WT tomato wild-type
  • ccos Cas9 mutated T1 lines were rinsed after 3 months of infestation with P. aegyptiaca seeds. Tubercles larger than 2 mm in diameter were considered for analysis.
  • Fig. 6 graphically depicts the average number of P. aegyptiaca tubercles and shoots attached to the mutant and non-mutant tomato plants in the pot assay.
  • the tomato host plant produces different kinds of strigolactones, mainly orobanchol, didehydroorobanchol isomer 1 and 2, and the aromatic strigolactone solanacols, including the recently identified orobanchyl acetate, 7-hydroxyorobanchol isomers 1 and 2, and 7-oxoorobanchol.
  • strigolactones a family of chemical molecules
  • Orobanche preferentially utilize orobanchol as the most active germination stimulant (>80% germination)
  • solanocol and 7-oxoorobanchol are weak stimulants.
  • CCD8 was modified in line 11a (see Table 1), and the plant exhibited the typical dwarfing and shoot-branching phenotypes of reduced strigolactone, its orobanchol content was higher than in the other modified lines. The higher orobanchol content was consistent with its lower resistance to P. aegyptiaca.
  • FIG. 7 graphically shows an analysis of orobanchol content in the WT roots as compared to tomato CCDS Cas9 edited lines (1, 2, 5 and 11) carried out by LC-MS/MS analysis ⁇ +SD n(2) ⁇ .
  • Fig. 8A presents a PCR product sequence alignment of CCDS Cas9 edited lines (1, 2, 5 and 11) used for the above LC-MS/MS analysis.
  • PAM protospacer adjacent motif
  • Bsrl site marked by a black triangle and shown in bold underlined
  • DNA deletions are shown in black dots and deletion sizes (nt) are marked on the right side of the sequences
  • Nucleotide sequence inside the red box encodes for stop codon.
  • Fig. 8B schematically depicting the amino acid sequences of line 2a (His-243 deletion) and line 11a (His-243 and Pro-244 deletion) of the present invention compared to wild-type (WT) CCD8 proteins.
  • CCDS Cas9 mutation altered the profile of different types of carotenoids and its derivative, such as total carotenoids (lutein; b-carotene were substantially altered from the wild type).
  • the expression of the prominent gene Phytoene desaturase-1 (Solyc03g 123760) and Lycopene cyclase l-b ( Solyc04g040190 ), involved in the carotenoid biosynthetic pathway which act upstream of CCD8 39 ’ 40 were analyzed.
  • Results obtained using quantitative real-time PCR demonstrated that expression of PDS1, LCY-b and CCD8 was upregulated in CCDS Cas9 edited T1 lines as compared to the wild type plants.
  • FIG. 9 showing the carotenoid contents in CCD8 Cas9 mutants and relative transcript expression of three candidate genes in CCD8 Cas9 mutated lines of the present invention.
  • Fig. 9B graphically quantifying the transcript levels of SICCD8, SIPDS1 (phytoene desaturase 1) and SlLCY-b (lycopene b-cyclase) in roots of ccos Cas9 edited lines and wild type (WT) plants using quantitative real-time PCR.
  • Expression level of transcript is displayed after normalization with internal control tomato elongation factor- la (EFl-a). Bar with different letter are significantly different from each other (student t-test, p ⁇ 0.05 when compared with the controls).
  • Results represent the average of three technical repeats + SE, showing fold increase in expression of transcripts relative to the control plants. Bars represent the average of pooled three plant roots from each line.
  • Fig. 11 depicting agarose gels displaying the identification of transgene (Cas9) free plants in T1 generation of two ccos Cas9 mutated lines of the present invention (line 2 as depicted in Fig. 11A, and line 5 as depicted in Fig. 11B).
  • the putative off-target sites associated with CCDS sgRNA were evaluated by CRISPR-P program using the CCDS sgRNA sequence against the tomato genome. Three potential off-targets sites with high scores, which occurred in the intergenic and CDS regions of the tomato genome were analyzed. Two plants from each line were selected from the T1 generations of CCDS Cas9-edited tomato plants. Sequencing of PCR products from these regions revealed no changes in the putative off-target sites in the ccos Cas9 -mutant lines.
  • Fig. 12 depicting a graphical presentation of PCR product sequence chromatogram of potential off-target of the CCDS sgRNA.
  • CCDS Cas9 mutant lines show highly branched shoots irrespective to the type of mutation but no significant differences were found in the root mass between ccos Cas9 mutated and control tomato plants.
  • Fig. 13A depicting characteristic shoot branching phenotype of CCDS Cas9 mutants (lines 1,2,5 and 11) compared to wild type plants. It is evident from the results that the ccos Cas9 tomato lines exhibit highly branched shoots.
  • Fig. 13B graphically presenting that there are no significant differences in the root mass of CCDS Cas9 mutants (lines 1,2,5 and 11) compared to wild type plants.

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Abstract

La présente invention concerne une plante de tomate présentant une résistance accrue aux mauvaises herbes parasitaires, et des procédés de génération de ladite plante. La résistance est obtenue par rupture et mutation des dioxygénases 8 de clivage de caroténoïde (CCDS) du gène de biosynthèse de strigolactone à l'aide du système CRISPR/Cas9. Outre la résistance aux mauvaises herbes parasitaires P. aegyptiaca, les plantes mutées sont caractérisées par des traits morphologiques uniques par rapport aux plantes de type sauvage.
PCT/IL2020/051289 2019-12-15 2020-12-15 Moyen et procédés de fourniture d'une résistance contre les mauvaises herbes parasitaires Ceased WO2021124323A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN119874861A (zh) * 2025-01-10 2025-04-25 中国科学院遗传与发育生物学研究所 SlABCG45基因在提高番茄的列当抗性和产量中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BARI, V. K. ET AL.: "CRISPR/Cas9-mediated mutagenesis of CAROTENOID CLEAVAGE DIOXYGENASE 8 in tomato provides resistance against the parasitic weed Phelipanche aegyptiaca", SCIENTIFIC REPORTS, vol. 9, no. 1, 7 August 2019 (2019-08-07), pages 1 - 12, XP055835958 *
KOHLEN, W. ET AL.: "The tomato CAROTENOID CLEAVAGE DIOXYGENASE 8 (S 1 CCD 8) regulates rhizosphere signaling, plant architecture and affects reproductive development through strigolactone biosynthesis", NEW PHYTOLOGIST, vol. 196, no. 2, 24 August 2012 (2012-08-24), pages 535 - 547, XP055203214 *
MALZAHN, A. ET AL.: "Plant genome editing with TALEN and CRISPR", CELL & BIOSCIENCE, vol. 7, no. 1, 24 April 2017 (2017-04-24), pages 1 - 18, XP002785201, DOI: 10.1186/s13578-017-0148-4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119874861A (zh) * 2025-01-10 2025-04-25 中国科学院遗传与发育生物学研究所 SlABCG45基因在提高番茄的列当抗性和产量中的应用

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