WO2023129099A1

WO2023129099A1 - Developing orobanche (orobanche spp., phelipanche spp.) resistance in tomato (solanum lycopersicum), using gene editing technology (crispr)

Info

Publication number: WO2023129099A1
Application number: PCT/TR2022/051703
Authority: WO
Inventors: Cansu BULBUL; Nedim MUTLU; Hasan PINAR; Inanc SOYLU
Original assignee: Akdeniz Universitesi; Erciyes Universitesi
Priority date: 2021-12-31
Filing date: 2022-12-30
Publication date: 2023-07-06

Abstract

The invention relates to the development of orobanche (Orobanche spp., Phelipanche spp.) resistance in tomato (Solanum lycopersicum) using gene editing technology (CRISPR) and to a method for the development of orobanche-resistant lines and CRISPR guide RNAs (sgRNA) designed for it. In the invention, with the CRISPR/Cas9 system designed to target the exon 1 and 3 regions of the CCD7 (SlCCD7) gene on the 1st chromosome of tomato, the in-frame mutation between Thr42-Val105 in the SlCCD7-exon 1 region of four T1 progenies is achieved, the deletion of 62 amino acids, resulting in orobanche resistant tomatoes without the undesirable phenotypic changes of prior art.

Description

DEVELOPING OROBANCHE (OROBANCHE SPP., PHELIPANCHE SPP.) RESISTANCE IN TOMATO (SOLANUM LYCOPERSICUM), USING GENE EDITING TECHNOLOGY (CRISPR)

Technical Field of the Invention

The invention relates to the development of resistance to orobanche (Orobanche spp., Phelipanche spp.) in tomato (Solarium lycopersicum) using gene editing technology (CRISPR) and to a method for the development of orobanche- resistant lines and CRISPR guide RNAs (sgRNA) designed for it.

State of the Art

Tomato is one of the major plants with economic value, which is produced and consumed intensively in the world. However, factors such as environmental conditions, soil structure, plant nutrition practices, irrigation methods, pathogens, and weeds are frequently encountered in tomato production, which limit the production and thus negatively affect the total yield and quality of the plant. One of the factors affecting the yield of tomatoes in the world and in Turkey is orobanches which are parasitic weeds (Phelipanche spp./Orobanchespp.).

Orobanche causes important yield and quality loss in cultivated plants such as eggplant, potato, tobacco, sunflower, broad bean, lentil, etc. including tomatoes in most of the countries. Orobanches are chlorophyll-free fully parasitic plants that can survive in the soil for a long time, produce thousands of seeds very small in size, and show host-dependent development. By absorbing the water and dissolved nutrients from the roots of the host plant with the suction organ it develops after germination, it provides its energy need, which it cannot obtain through photosynthesis. On the other hand, quality and yield losses are observed in the host plant. The seeds of the orobanche surprisingly also germinate through the host plant under suitable environmental conditions. Seeds germinate by means of "Strigolactone", a plant hormone secreted from the host plant roots and synthesized by the carotenoid pathway. It is known that genes named CCD7 and CCD8 (Carotenoid Cleavage Dioxygenase 7 and 8), which are involved in the first steps of carotenoid synthesis in tomato, play a fundamental role in the production of strigolactone. With the production of strigolactones, a signalling mechanism is formed that triggers the germination of parasitic orobanche.

During efficient genetic transformation of plants with the gene of interest, certain selectable marker genes, i.e. , markers, are also used to identify transgenic plant cells or tissues. The presence of selectable marker genes in transgenic crops can be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, rendering the pests resistant to treatment with herbicides or antibiotics, respectively. The future potential of transgenic technologies for crop improvement largely depends on designing the predictably stable expression of multiple transgenic traits and preventing the transfer of unwanted transgenic material to non-transgenic crops and related species. Therefore, the production of transgenic plants without an effective indicator/marker is now a critical requirement for their commercial distribution as well as engineering multiple and complex traits. For this reason, it is of great importance to develop marker-free transgenic plants in the process of providing resistance to orobanche.

In the prior art, SI-ORT1 mutant was obtained by the method of fast neutron (FN) mutagenesis of the tomato variety M-82 by Dor et al. [1 ], Although it was determined that this mutant was resistant to three different Orobanche spp species (Phelipanche ramosa, Orobanche cernua, and Orobanche crenate) , it was determined that the phenotypic changes previously determined by Koltai et al. were also present in the plant. Due to the negative effect of strigolactone deficiency in mutant plant on auxin (plant hormone) transport, an increase in shoot number (bushy appearance) and therefore a change in shoot/root ratio compared to WT plant (not mutated). In the prior art, the SICCD8 gene, which is found in tomatoes and plays a role in strigolactone synthesis, was silenced by RNAi (RNA interference)-mediated method by Kohlen et al. [2], Although 90% resistance was achieved against Phelipanche ramosa, arbuscular mycorrhizal symbiosis, apical dormancy and fruit yield were only slightly affected in the resulting mutants. In addition to these, an increase in the number of shoots in the plant, decrease in plant height (dwarfism), increase in adventitious root and node formation, smaller flower and fruit size and less number and size of seed formation per fruit occurred. In addition, the obtained orobanche resistant plant is classified as GMO since it contains the foreign gene (transgene).

Patent application WO2021124323A1 in the prior art describes a tomato plant that exhibits resistance to parasitic weeds. The resistance described here is achieved by cleaving and mutating the strigolactone-biosynthesis gene carotenoid cleavage dioxygenases 8 (CCD8) using the CRISPR/Cas9 system. In addition to resistance to the parasitic weed P. aegyptiaca, mutated plants were noted to exhibit unique morphological features compared to wild-type plants. It is noted that the term "CCD8-Cas9 knockout phenotype" as used herein refers to any phenotype exhibited by tomato plants regulated by CRISPR-Cas9; and that this phenotype may manifest as stunting, excessive shoot branching, incidental root formation, increase in lateral roots, decrease in fruit size, decrease in orobanche content, increase in total carotenoid level, or any combination of these.

In the study of Bari et al., who are one of the same inventors and on a similar subject to the patent described above, different mutations (different deletion and insertions (nucleotide (nt) deletion or insertion)) were created in the 2^nd exon of the SICCD8 gene with a single sgRNA (5'-TTCATTCAGCTCATCCAGTGG-3' sequence) designed within the scope of the CRISPR/Cas9 method [3], As a result of sequencing in TO lines where mutations were obtained, 1 nt deletion in L1 , 3 nt deletions in L2, and biallelic mutations in L5 and L11 (4 different mutations occurred: 6, 5 or 4 nt deletion, or 1 base insertion) were found to exist. T1 generation was created by the inbreeding (selfing) of TO lines, and the mutations were heritable. Additional sequencing at T1 revealed a homozygous mutation (with the same mutation in both alleles) in L1 and L2. Although resistance to Phelipanche aegyptiaca was obtained due to the decrease in strigolactone level in mutant lines (higher resistance was obtained in L1 , L2, L5 compared to L11 ). morphological changes such as increased carotenoid level, dwarfism, excessive shoot branching and adventitious root formation occurred in all mutants. In addition, although more fruit per plant was obtained due to the increase in the number of shoots, a decrease in fruit size was detected.

In another study by Butt et al., in the prior art, CCD7 mutation with CRISPR/Cas9 is mentioned, but the study here is conducted on rice [4], As mentioned herein, reduction of strigolactone (SL) production in the prior art is known to show promise in plant species including rice, peas, broad bean (Vicia faba), tomato (Solarium lycopersicum) and maize (Zea mays). However, significant reductions in SL levels may compromise other factors such as the formation of arbuscular mycorrhizal symbiosis and therefore lead to inefficient productions. There is a need to develop gene editing technologies that enable the reduction of strigolactone (SL) production without causing undesirable phenotypic characteristics to overcome the drawbacks of the prior art.

In the prior art, there is no manipulation with CRISPR/Cas9 for the SICCD7 gene, which is involved in the early stages of the strigolactone pathway. However, in genes such as SICCD8 and Slmaxl, lines with undesirable agromorphological characteristics in terms of plant breeding were common in all mutants obtained by other methods (such as FN, TILLING, RNAi) including CRISPR/Cas9 or SICCD7. These features can be listed as multi-headedness due to shoot branching, bushy appearance, fertilization problems due to deterioration in flower/fruit characteristics, and deterioration in shoot/root ratio.

In addition, in studies in which homozygous biallelic mutations were created by different CRISPR/Cas9 methods, antibiotic resistance and marker-free mutants free of Cas9 transgenes could not be obtained. In addition, in the studies in the prior art, an “in-frame” mutation that causes a large-scale deletion could not be obtained with the CRISPR/Cas9 method, and mutations resulting in frame shift occurred in the open reading frame in general. Prior art studies, in which mutations resulting in frame shifts in the open reading frame occurred, caused such changes in phenotypic properties as a result of the disruption of interactions with other proteins due to the complete destruction of the functions of the proteins.

Due to the occurrence of undesirable agromorphological features in terms of plant breeding in mutants obtained in the methods of developing resistance against orobanche using gene editing technology, which is included in the previous art, and because the lines in which resistance is developed are transgenic plants containing selectable markers; while developing resistance to orobanche, there is a need to develop methods that do not cause undesirable agromorphological characteristics in mutants for plant breeding, and that provide marker-free lines (no antibiotic or herbicide resistance) and free of Cas9 transgenes, and new guide RNAs to be used in these methods.

Brief Description of the Invention

In the invention, with the CRISPR/Cas9 system designed to target the exon 1 and 3 regions of the CCD7 (SICCD7) gene on the 1^st chromosome of tomato, the inframe mutation between Thr42-Val105 in the native S/CCD7-exon 1 region of four T1 progenies is achieved, resulting in the deletion of 62 amino acids, resulting in marker-free, orobanche resistant tomatoes without the undesirable phenotypic changes of prior art. Also, mutation between Thr42-Val105 in the native SICCD7- exon 1 region, which is formed between Thr42-Val105 in the natural SICCD7- exon 1 region with the SEQ ID NO:3 sequence and causes in-frame mutation with the deletion of 186 bp (62 amino acids), can be transferred to any desired tomato genotype by "marker assisted back-crossing" method, using the primer pairsS/CCD7-sgRNA1 -F has SEQ ID NO:5 sequence and S/CCD7-sgRNA1 -R has SEQ ID NO:6 sequence as molecular markers in order to select for the modified allele. The first aim of the invention is to provide a method that does not cause undesirable agromorphological and phenotypic characteristics in terms of plant breeding in mutants while improving resistance to orobanche (Orobanche/Phelipanche spp.) in tomato. By means of the use of CRISPR/Cas9 technology and designed guide RNAs in tomato in the invention, in the T1 generation, which is obtained by inbreeding of the TO line determined to carry a heterozygous biallelic mutation (18 nt + 186 nt deletion) in the SICCD7 gene, which is involved in the strigolactone (SL) pathway, four each of T1 lines with homozygous biallelic mutations (18 nt deletion or 186 nt deletion) are obtained as a result of the segregation of this inherited mutation. With this homozygous biallelic mutation occurring in the exon 1 region of the SICCD7 gene, a large protein fragment corresponding to 62 amino acids (the region between Thr42- Val105) is deleted as a result of the deletion of 186 nt. However, since this deletion is a multiple of three, the mutation did not cause a "frame shift" and was described as an "in-frame" mutation. This may cause various molecular changes, including changes in protein-DNA binding sites, due to the alteration of the secondary and tertiary structure of the protein, but it does not cause undesirable phenotypic features as reported for frame shift mutations.

In the invention, resistance against Orobanche/Phelipanche spp. is obtained in tomato by in-frame mutation. Moreover, apart from the partial shortening of the internodes in the obtained Orobanche/Phelipanche spp resistant mutant lines, no agronomic features (bushiness associated with an increase in the number of shoots, fertilization problems due to deterioration in flower/fruit characteristics, deterioration in shoot/root ratio) that are observed in the SL-knock-out mutant lines in other studies in the literature and are undesirable in terms of plant breeding are not detected. For this reason, these mutants obtained are of great commercial importance, and these resistant lines are considered as important breeding materials in terms of their agronomic characteristics.

Different from the patent application WO2021124323A1 in the prior art and the studies of Bari, 2 sgRNAs of CCD7 and CCD8 were transformed into tomato, but only CCD7-sgRNA1 was transferred as a result of the analysis. As a result, with the invention, a mutation was created in the CCD7 gene, which was found before CCD8 gene in the pathway. This mutation also prevented the undesirable phenotype results in the prior art. Thus, a mutant plant with intact agronomic properties was obtained with the invention.

Another aim of the invention is to obtain tomatoes that are free of antibiotic resistance and Cas9 transgenes, do not contain markers and do not belong to the class of GMO (genetically modified organism) while obtaining resistance to orobanche. It has been determined that some of the in-frame mutant lines (C9, C22) provided by the invention are free of Cas9 and antibiotic resistance genes, are marker-free plants, that is, they are not GMOs and are completely safe for consumption. By means of the segregation of Cas9 and antibiotic resistance- related transgenes in two T1 lines carrying this homozygous mutation, it is seen that these plants do not contain any extra gene (that they are marker-free) apart from the plant gene. The mutation that provides orobanche resistance in C9 and C22 lines within the scope of the invention can be transferred to any desired tomato varieties by classical or marker assisted backcrossing and yield losses due to orobanche damage can be reduced.

An additional advantage obtained indirectly by means of the invention is the elimination of the economic, environmental and health hazards caused by the chemical control methods used against these parasitic weeds. In addition, the methods used within the scope of the invention have the potential to be used in other crop plants that lose yield due to orobanche.

By means of the invention; the guide RNAs (sgRNA), designed to target exon 1 and 3 regions of the CCD7 (SICCD7) gene on the 1 st chromosome of tomato, were integrated into a binary vector with the dicotyledon-specific codon optimized Cas9 gene, and its expression in tomato was achieved by Agrobacterium- mediated plant transformation. Therefore, as a result of successful manipulation within the scope of the invention with the CRISPR/Cas9 system, it is seen that this mutation occurring in the relevant natural S/CCD7-exon 1 region negatively affects the strigolactone pathway and provides resistance against Orobanche/Phelipanche spp. With the invention, while improving resistance to orobanche, a method that does not cause undesirable agromorphological characteristics in terms of plant breeding in mutants and that enables obtaining lines free of antibiotic resistance and Cas9 transgenes, and guide RNAs for use in this method are provided.

Description of Drawings

Figure 1 : Representative map showing the exons targeted by sgRNAs on the SICCD7 gene.

Figure 2: Representative image of the designed sgRNA 1 and sgRNA 2 cassettes (RE 1 : Pad (5'...TTAAT/TAA...3'), RE 2: Acll (5'...AA/CGTT...3')).

Figure 3: Different PCR assays obtained with DNA of C36-T0 a) UV imaging of the 638 bp band obtained from the amplification of C36-T0 DNA with the BASTA F/R primer pair; b) UV imaging of the 582 bp band obtained from the amplification of C36-T0 DNA with the pcoCas9-F/R primer pair; c) UV image of the sequenced bands obtained from the amplification of C36-T0 DNA with primer pair SICCD7- sgRNA1-F/R (K) and S/CCD7-sgRNA2-F/R (L).

Figure 4: PCR assays at T1 generation using DNA extracted from 23 C36-T1 progenies (a) PCR products (638 bp) obtained using the BASTA F/R primer pair; (b) PCR products (754 bp) obtained using the pcoCas9-F/R primer pair (M: DNA marker).

Figure 5: Chromatograms obtained by sequencing the PCR product of C36-T0 using the S/CCD7-sgRNA1 -F/R primer by Sanger method.

Figure 6: DNAs extracted from 23 C36-T1 progenies and PCR products obtained using primer pair S/CCD7-sgRNA1 -F/R (K: 267-F1 (WT/unmutated) used as control, M: DNA marker).

Detailed Description of the Invention

The invention relates to the development of orobanche (Orobanche spp., Phelipanche spp.) resistance in tomato (Solanum lycopersicum) using gene editing technology (CRISPR) and to a method for the development of orobanche- resistant lines and CRISPR guide RNAs (sgRNA) designed for it. Also, mutation between Thr42-Val105 in the native S/CCD7-exon 1 region, which is formed between Thr42-Val105 in the natural SICCD7-exon 1 region with the SEQ ID NO:3 sequence and causes in-frame mutation with the deletion of 186 bp (62 amino acids), can be transferred to any desired tomato genotype by "marker assisted back-crossing" method, using the primer pairs S/CCD7-sgRNA1 -F has SEQ ID NO:5 sequence and S/CCD7-sgRNA1-R has SEQ ID NO:6 sequence as primers for the molecular marker- in order to select for the modified allele.

Within the scope of the invention, CRISPR guide RNAs called sgRNAI designed to target the exon 1 region of the CCD7 (SICCD7) gene on the 1^st chromosome of the tomato and sgRNA2 designed to target the exon 3 region of the CCD7 (SICCD7) gene are used. This CRISPR/Cas9 system provides an in-frame mutation between Thr42-Val105 in the S/CCD7-exon 1 region of four T1 progeny and causes deletion of 62 amino acids. The designed and used sgRNA2 targets exon 3, but out of these two sgRNAs (sgRNAI and sgRNA2), only sgRNAI acted functionally and caused a mutation in the exon 1 region it is associated with. crRNA (target) and tracrRNA (backbone) are needed for editing with the CRISPR/Cas9 system to be used in in-vivo conditions. Therefore, the synthetically designed sgRNA complex (crRNA+tracRNA) is sufficient for the Cas9 CRISPR/Cas9 system to work. These sgRNAs consist of a target sequence (ideally 20 base pairs) and a PAM sequence (NGG for SpCas9), which may vary depending on the microorganism that is the source of the Cas9 used. When expressed together with Cas9 in the cell, sgRNAs with the appropriate promoter and terminator sequence added form a double-stranded break (DSB) by cutting the double-stranded structure between the -3^th and -4^th base pairs found before the PAM sequence on the DNA that is complementary to the target sequence. In this context, the sequence information (Gene ID: 100313501 ) of the tomato CCD7 (SICCD7) gene obtained from the NCBI/Gene database was processed into the CRISPRDirect program and sgRNA (sgRNAI and sgRNA2) designs were made. In addition, the sgRNAs obtained through this program have optimized properties and also identify regions with off-target potential. In this way, 100% targeted gene editing can be achieved. Two sgRNAs targeting different exon regions of the gene were designed to increase the probability of mutation in the gene of interest. Matching AtU6-26 promoter and terminator sequences were added to these sgRNAs with optimum properties in dicot plants, allowing the expression of the first (sgRNAI ) to exon 1 region and the second (sgRNA2) to exon 3 (Figure 1 ). Due to the long sequences of these two sgRNAs, it was predicted that they would be synthesized in two parts, and Acll and Pad enzyme restriction sites were added to the 5’ head and 3’ end parts of the sgRNAs to be combined in a molecular cloning (Figure 2).

Rationally designed sgRNA sequences for the SICCD7 gene are provided in the sequence listing. The first three nucleotide (nt) region in the sequence list is the sequence of the PAM sequence (CCA). The part that was synthesized and used in the study is the 20 nt target sequence, excluding the PAM sequence. Table 1 contains details of S/CCD7-sgRNA1 (SEQ ID NO: 1 ) and sgRNA2 (SEQ ID NO:2).

Table 1. SICCD7 sgRNAI and SICCD7 sgRNA2 info.

The synthesized sgRNAs were excised from the pTZ57R vectors with the help of appropriate enzymes, and the two sgRNAs were ligated in one piece to the pFGC-pcoCas9 (Addgene #52256) transformation vector carrying the components of the CRISPR/Cas9 system with the help of NEB, T4 ligase enzyme. At this stage, sgRNAs with Pad restriction sites at their ends were attached to the pFGC-pcoCas9 vector, which was linearized from the Pad restriction site it contains, and a new modified plasmid (pFGC- pcoCas9s/ccDzsgRNAi+2) was obtained. It was ensured that the sgRNAs linked to each other at their other ends via the Acll restriction sites were included in the pFGC-pcoCas9 vector as a single piece.

In order to increase the probability of mutation in the invention, it was preferred to use sgRNA pair, namely two sgRNAs (sgRNAI and sgRNA2). In the native S/CCD7-exon 1 region with the sequence SEQ ID NO:3, first sgRNA (sgRNAI ) causing in-frame mutation by deletion of 62 amino acids occurring between Thr42-Val105 has the sequence SEQ ID NO:1 targeting the exon 1 region of the native SICCD7 gene, and the second sgRNA (sgRNA2) has the sequence SEQ ID NO:2 targeting the exon 3 region of the native SICCD7 gene. However, sgRNAI can also be used alone, as it is ultimately understood that only SICCD7- sgRNAI causes the mutation.

After being cloned and validated using E. coli, DH5a strain and the standard heatshock method for bacterial selection, pFGC-pcoCas9s/ccD7sgRNAi+2 transformation vector with kanamycin (antibiotic) resistance (colonies selected from media containing kanamycin selection were confirmed by PCR and sequencing) were transferred to Agrobacterium tumefaciens EHA105 strain, which is frequently used in plant transformation in tomato, by electroporation (2500V, 25 pF capacitance and 400 ohm) method. Transformant Agrobacterium strains (A.tumefadensEHM05-pFGc-pcocas9-siccD7) were prepared in this way.

In PCR analyses, DNAs extracted according to the 2% CTAB protocol were amplified with the relevant primer pairs in the sequence list, and all analyses were performed in accordance with the protocol of the Taq polymerase kit. The primers used within the scope of the invention are explained in detail below. As PCR condition, the protocol of “initial denaturation at 95°C, denaturation at 95°C for 45 seconds for 35 cycles, annealing at the appropriate temperature (Tm suitable for the primer sequence used) for 45 seconds; extension at 72°C for 45 seconds, and a final extension at 72°C for 10 minutes” was used. The PCR products obtained under the specified conditions were run on a gel containing 1.5% (by mass) agarose and 0.005% ethidium bromide, at a potential difference of 100V, for 45 minutes. After electrophoresis, the agarose gel was visualized in the UV imaging system.

In plant transformation and tissue culture studies, the "tomato transformation" protocol known in the prior art was applied. This protocol consists of six basic stages: seed germination, pre-infection, infection, shooting, rooting and acclimatization.

In the first step, which is the seed germination step, the seeds are germinated by keeping them in the germination medium for 10 days after the sterilization process. Within the scope of this study, seeds of indeterminate tomato genotype 267-F1 obtained from Proto Seed, Antalya were used. However, in general, all tomatoes of the genus Solanum lycopersicum can be gene-edited with the sgRNAs disclosed in the invention. In the second stage, explants are obtained by cutting the cotyledon leaves formed as a result of germination before infection. In the explants obtained, scar tissue is formed with the help of a scalpel, and Agrobacterium infection is triggered at the infection stage.

In the third stage of infection, explants and A. tumefaciens bacterial cultures carrying transformation vectors (Atumefac/ensEHAio5-pFGc-pcocas9-s/ccDz) are brought together for 30 minutes and the bacteria are allowed to infect the plant tissue. In the fourth stage, which is shooting, shoots are formed by regeneration in a medium containing selection from transformant plant cells at appropriate concentration (for this study, the selection of BASTA (with the “bar” (BASTA) resistance gene on the transformation vector) is used, which is an herbicide containing 2 mg/ml glufosinate-ammonium) and Zeatin (cytokinin hormone). In resistant plant tissue, it contains the vector EHA105-pFGC-pcoCas9-S/CCD7, which contains guide RNAs of SICCD7 (sgRNAI alone or sgRNA2 together with sgRNAI ). In the fifth stage, which is rooting, root formation is provided from the shoots transferred to auxin-containing media. In the sixth and final stage of acclimatization, transgenic plants, the upper shoot and lower root parts of which are formed, are transferred to the soil from the rooting media and their adaptation to natural environmental conditions is ensured. CCD7 exon 1 of the resistant plant obtained within the scope of the invention has the sequence SEQ ID NO:4 and differs from the natural S/CCD7-Exon1 sequence with the sequence SEQ ID NO:3. As a result of the gene editing described in the invention, 186 nucleotides (nt) are deleted with the homozygous bial lei ic mutation occurring in the exon 1 region of the natural SICCD7 gene. The native Solanum lycopersicum CCD7-exon1 sequence with SEQ ID NO:3 is 817 nt length, while the SICCD7 mutant-exonl sequence with SEQ ID NO:4 is 631 nt length. As a result of this change, a large protein fragment (the region between Thr42-Val105) corresponding to 62 amino acids is deleted.

Transgenic tomato (Solanum lycopersicum) plant resistant to orobanche (Orobanche spp., Phelipanche spp.) obtained within the scope of the invention comprises a vector having the Cas9 gene containing the first guide RNA with the sequence SEQ ID NO:1 targeting the exon 1 region of the native SICCD7 gene in TO progeny or a vector having the Cas9 gene containing the first guide RNA having the sequence SEQ ID NO:1 , together with the second guide RNA having the sequence SEQ ID NO:2 targeting the exon 3 region of the native SICCD7 gene.

By means of the aforementioned guide RNA or guide RNA pair, deletion occurs in the SICCD7-exon 1 region, and the resulting transgenic tomatoes, has SEQ ID NO:4 of SICCD7-exon 1 region. These transgenic tomatoes can be transferred to the desired line or variety by backcrossing method, thus providing the line or variety with orobanche resistance.

In the determination of transgenic plants and in field studies, analyses were performed with TO plant DNAs extracted from plants obtained by transferring A. tumefadensEHM05-pFGc-pcocas9-siccD7 vector to 267-F1 indeterminate type tomato and regeneration in medium containing BASTA selection. All primers described in the invention are primers designed within the scope of the invention and are used for verification purposes (Table 2). The primers used within the scope of the invention are: • S/CCD7-sgRNA1 forward (F)/reverse (R) primers as primers that amplify the portion containing sgRNAI targeting the exon 1 region of the SICCD7 gene,

• S/CCD7-sgRNA2 forward (F)/reverse (R) primers as primers that amplify the portion containing sgRNA2 targeting the exon 1 region of the SICCD7 gene,

• S/CCD7-forward (F)/reverse (R) primers as primers amplifying the SICCD7 gene,

• BASTA-forward (F)/reverse (R) primers that amplify the “bar” resistance gene on the transformation vector,

• pcoCas9- forward (F)/reverse (R) primers that amplify the promoter region of the pcoCas9 gene

Table 2. The info on used primers

First, it was determined that a successful transformation occurred in the C36-T0 line, which produced the correct size band at 638 bp (Figure 3a) by PCR amplification with the BASTA F/R primer pair designed to target the bar resistance gene (selectable marker) on the A. tumefadensEHM05-pFGc-pcocas9-siccD7 vector (BASTA analysis). The examples called C1 -36 mentioned within the scope of the invention represent different trials and the plant belonging to this trial in order not to mix the plants. To confirm this result, the correct band of 754 bp was obtained as a result of PCR amplification with the S/CCD7-sgRNA-F/R primer pair, which was designed to target sgRNAs carried on the same vector (Figure 3b).

In order to determine the mutation status in C36-T0, PCR reaction was performed with primer pairs (S/CCD7-sgRNA1 -F/R and S/CCD7-sgRNA2-F/R) designed to target sgRNAI and sgRNA2 in exon 1 and 3 regions of the SICCD7 gene. As a result of electrophoresis of PCR products, the bands in Figure 3c were obtained and it was determined that a large mutation (deletion (base deletion)) occurred in the exon 1 region targeted by S/CCD7-sgRNA1 . The resulting PCR products were cleaned from the gel in separate pieces and sequenced. As a result of bioinformatic studies performed using chromatograms obtained as a result of sequencing, it was determined that there is a potentially biallelic heterozygous - 18 and -186 nt deletion only in the sgRNAI region of the C36-T0 plant (Figure 5). In Figure 5, the sgRNAI region after the Cas9 cleavage site was partially found in the allele with -18 nt deletion compared to the original sequence, while the sgRNAI region was completely deleted in the other allele with -186 nt deletion. The resulting resistant plants (TO progeny) contained sgRNAI and sgRNA2 of CCD7 (SICCD7). As a result of the experiments, it was determined that sgRNAs for CCD8 was not transferred to the plant genome (C36 plant genome). As a result of the sequencing performed in the same way, it was understood that the mutation in C36 and its progenies was caused by the operation of only sgRNAI designed for CCD7.

In order to understand the heritability of this heterozygous mutation, to resequence the mutation regions and to make phenotypic observations in C36- T 1 lines with different allelic status, 23 T 1 progenies were obtained by inbreeding C36-T0 plant. Testing with the BASTA F/R primer pair at T1 using DNA extracted from 23 C36-T1 progenies was performed in the same way (Figure 4a). Here, PCR products were electrophoresed on a 1.5% agarose gel at 100 V for 45 minutes in the presence of GeneRuler 1 kb DNA marker. As an additional confirmation, testing with the pcoCas9-F/R primer pair designed from the promoter region of the pcoCas9 gene on the Afumefac/ensEHAi05-pFGc-pcocas9- S/CCDZ vector (Figure 4b) yielded results that exactly matched up with the BASTA analysis.

Based on the image in Figure 4a and Figure 4b, it was determined that there were no bar or Cas9 transgenes in T1 progenies C9 and C22, therefore these plants did not contain markers. Again, testing was performed with the S/CCD7-sgRNA1 F/R primer pair to determine the inheritance and segregation of the mutation in the sgRNAI region, which was made with the same T1 DNAs and determined to have a mutation in C36-T0, and to resequence the alleles carrying the mutation. It was determined that in 23 mutants obtained in this generation, 14 heterozygous biallelic mutations (-18 nt + -186 nt deletion), 5 homozygous biallelic mutations (- 18 nt deletion) and 4 homozygous biallelic mutations with -186 nt deletion were inherited. It was determined that the segregation fitted the expected 1 :2:1 Mendelian segregation (p < .05 (X² = 1.16)) and there was no new/different mutation. With the bioinformatic analysis of the chromatograms obtained by resequencing the upper and lower bands cleared from the gel, the exact same chromatograms of the heterozygous biallelic mutation determined in C36-T0 were obtained.

C9 and C22 are called non-transgenic and marker-free because Cas9, which is involved in the CRISPR-Cas9 system, has been segregated out from the plant genome in T1 progeny. The fact that these progenies do not contain transgene means that there is no longer any protein in their genome that would cut the double helix involved in the CRISPR-Cas9 system. For this reason, there is no longer any possibility of a new mutation occurring in their following generations. In other words, in some of the T1 progenitors, sgRNAI and sgRNA2 are separated from the plant genome and SICCD7 exon 1 of the resistant transgenic tomatoes obtained by gene editing described in the invention has the sequence SEQ ID NO:4.

The large deletion occurring between Thr42-Val105 in the native S/CCD7-exon 1 region of these 4 (C1 , C8, C9, C22) T1 progeny and causing deletion of 62 amino acids was evaluated as "homozygous "in-frame" mutation since it does not cause a frame shift mutation (due to a multiple of 3 bases deleted) The aforementioned in-frame mutation increases gene expressions in the carotenoid pathway and increases carotenoid levels, but it does not cause undesirable agronomic or morphologic properties.

It was observed that phenotypic changes consistent with the carried mutation status occurred as a result of periodic observations made with the cultivation of 23 T1 progeny under equal and suitable greenhouse conditions. Mutants with homozygous -186 nt deletion had shortening in plant height compared to the others. Mutant plants with -186 nt deletion were found to have the shortest internodes, while those with -18 nt deletion had the longest length and those with heterozygous mutations (-186 nt + -18 nt deletion) had intermediate internode lengths.

Compared to plants with homozygous biallelic -18 nt deletion, the internode length is the shortest in plants with homozygous biallelic -186 nt deletion, while the internode length is medium in plants with heterozygous biallelic mutations (- 18 nt + -186 nt). Mutant plants with -186 nt deletion had the shortest internode, while those with -18 nt deletion had the longest internode, and those with heterozygous mutations (-186 nt + -18 nt deletion) had intermediate length between the nodes. Plants with 186 nt deletion are still single stemmed, and no difference was observed in terms of agronomic characteristics such as fruit size and quality. References

1. Dor, E., Alperin, B., Wininger, S., Ben-Dor, B., Somvanshi, V. S., Koltai, H., Kapulnik, Y. & Hershenhorn, J. (2010). Characterization of a novel tomato mutant resistant to the weedy parasites Orobanche and Phelipanche spp. Euphytica, 171 (3), 371 -380.

2. Kohlen, W., Charnikhova, T., Lammers, M., Pollina, T., Toth, P., Haider, I., Pozo, M. J., Maagd, R. A. de, Ruyter-Spira, C., Bouwmeester, H. J. & Lopez- Raez, J. A. (2012). The tomato CAROTENOID CLEAVAGE DIOXYGENASE 8 (SICCD8) regulates rhizosphere signaling, plant architecture and affects reproductive development through strigolactone biosynthesis. New Phytologist 196(2), 535-547.

3. Bari, V.K., Nassar, J. A., Kheredin, S.M. et al. CRISPR/Cas9-mediated mutagenesis of CAROTENOID CLEAVAGE DIOXYGENASE 8 in tomato provides resistance against the parasitic weed Phelipanche aegyptiaca. Sci Rep 9, 11438 (2019).

4. Butt, H., Jamil, M., Wang, J.Y. et al. Engineering plant architecture via CRISPR/Cas9-mediated alteration of strigolactone biosynthesis. BMC Plant Biol 18, 174 (2018).

Claims

CLAIMS 1. Gene editing technology guide RNA (sgRNA) for use in developing resistance to orobanche (Orobanche spp., Phelipanche spp.) in tomato (Solarium lycopersicum), comprising the sequence SEQ ID NO:1 targeting the exon 1 region of the native SICCD7 gene, which occurs between Thr42-Val105 in the native SICCD7-exon 1 region and causes in-frame mutation with deletion of 62 amino acids. 2. Gene editing technology guide RNA (sgRNA) pair for use in developing resistance to orobanche (Orobanche spp., Phelipanche spp.) in tomato (Solanum lycopersicum), wherein the first sgRNA that occurs between Thr42-Val105 in the native SICCD7-exon 1 region with the sequence SEQ ID NO:3 and causes in-frame mutation with the deletion of 62 amino acids, has the sequence of SEQ ID NO:1 targeting the exon 1 region of the native SICCD7 gene and the second sgRNA has the sequence SEQ ID NO:2 targeting the exon 3 region of the native SICCD7 gene. 3. A transgenic tomato (Solanum lycopersicum) resistant to orobanche (Orobanche spp., Phelipanche spp.), comprising a vector having the Cas9 gene containing the first guide RNA with the sequence SEQ ID NO: 1 targeting the exon 1 region of the native SICCD7 gene that occurs between Thr42-Val105 in the native SICCD7-exon 1 region with the sequence SEQ ID NO:3 and causes an in-frame mutation by deletion of 62 amino acids, or a vector having the Cas9 gene containing the first guide RNA having the sequence SEQ ID NO:1 , together with the second guide RNA having the sequence SEQ ID NO:2 targeting the exon 3 region of the native SICCD7 gene. 4. A transgenic tomato (Solanum lycopersicum) resistant to orobanche (Orobanche spp., Phelipanche spp.), wherein SICCD7-exon 1 region has SEQ ID NO:4 sequence. Transgenic tomato (Solatium lycopersicum) according to Claim 4, wherein the tomato does not comprise Cas9 and bar transgenes. A mutation for use in selection of the modified allele with the marker assisted back-crossing method, which is formed between Thr42-Val105 in the natural SICCD7-exon 1 region with the SEQ ID NO:3 sequence and causes in-frame mutation with the deletion of 186 bp (62 amino acids), using the primer pairs S/CCD7-sgRNA1-F has SEQ ID NO:5 sequence and S/CCD7-sgRNA1-R has SEQ ID NO:6 sequence.