Development of drought-resistant tomato by altering root system and stomatal density

Abstract

The invention relates to the production of a drought-resistant genetically modified tomato by silencing Solyc01g080540 with SEQ ID NO:9 sequence and Solyc06084410 with SEQ ID NO:12 sequence from the AHP family involved in cytokinin signalling in tomato (Solarium lycopersicum) by the CRISPR / Cas9 gene editing method. In the said CRISPR / Cas9 system, gRNA 3 with nucleotide sequence SEQ ID NO:1, gRNA 25 with nucleotide sequence SEQ ID NO:2, gRNA 29 with nucleotide sequence SEQ ID NO:3 and gRNA 78 with nucleotide sequence SEQ ID NO:4 are used to target Solyc01g080540 and Solyc06084410 genes and to create mutations. The mutant tomato lines subject to the invention show a drought-resistant phenotype with physiological characteristics such as decreased stomatal number, increased root length and lateral root number.

Description

[0001] DEVELOPMENT OF DROUGHT-RESISTANT TOMATO BY ALTERING ROOT SYSTEM AND STOMATAL DENSITY

[0002] Technical Field Related to the Invention

[0003] The invention relates to a genetically modified tomato (Solatium lycopersicum), obtained by mutating and silencing the Solyc01g080540 and Solyc06084410 genes in tomato using gene editing technology (CRISPR), which shows drought resistance with the development of the root system and the decrease in the number of stomata, a method for producing this tomato, and guide RNAs (gRNA) to be used in this method.

[0004] State of the Art

[0005] Global warming is expected to increase temperatures, create a water crisis and increase the amount of carbon dioxide (CO2) in the planet's atmosphere in the next 30 years. In this respect, making plant species essential to human nutrition tolerant to these conditions is especially important. Especially with increasing temperatures and water crises, drought is becoming a problem. The development of plants is disrupted, and agriculture is adversely affected by drought.

[0006] Although methods such as drip irrigation systems, enriching the soil, and increasing the variety of crops planted are used against this drought challenge, these do not provide a permanent solution to the problem and create additional costs. Increasing drought resistance by targeting the plant, which is the subject of the problem, is a solution approach that does not cause continuous expenditures and is permanent. Therefore, producing plants resistant to drought is necessary for efficient agriculture suitable for drought conditions.

[0007] Resistance to drought conditions in plants is shown by certain physiological adaptations. For example, a well-developed root system is important for plants under drought conditions as it increases access to the scarce water content in the soil. On the other hand, the response of plants to environmental factors in terms of stomata also varies [1]. Plants take in the CO2 they need for photosynthesis through stomata. However, only CO2 is insufficient for photosynthesis, and water must be supplied to them. With the increasing amount of CO2 and decreasing amount of water in the coming years, plants with intensive stomatai development will be negatively affected by this situation and more affected by drought conditions. On the other hand, plants with reduced stomatai numbers have an advantage in these conditions compared to both plants with reduced stomatai spacing and plants with unchanged stomatai numbers [2],

[0008] Among the methods aiming to create the phenotypes of the species mentioned above, genetic intervention in the plant to create the desired adaptation is at the forefront. With CRISPR / Cas9 gene editing technology and developments involving this technology, it has become easier to perform mutations in specific regions, and this technology has started to be preferred more frequently in genetic interventions. In CRISPR / Cas9 technology, the Cas9 endonuclease enzyme acts on genetic material by cutting the target DNA region. This technology uses specially designed guide RNAs (gRNAs) to identify the targeted DNA region and ensure that the Cas9 enzyme binds only to this target region. [3]

[0009] In order to produce tolerant plants through such an intervention, the genes responsible for the stress condition to be tolerated must first be identified. After identifying the genes that negatively affect tolerance to stress conditions, the target genes are silenced using a vector that will create mutations specific only to these genes using the CRISPR / Cas9 gene editing method.

[0010] AHP (Arabidopsis thaliana histidine phosphotransfer protein) genes, which are responsible for the signalling of the growth-related hormone called cytokinin in studies with Arabidopsis thaliana, which is used as a model organism in plants, also play a role in drought stress responses in Arabidopsis thaliana plants. However, it is important to conduct separate studies for each plant species to determine the role of the genes. In the patent application CN110669785A in the prior art, the gene encoding the SILOB40 protein is silenced to prevent the negative control responses of the LOB protein family against drought in tomato plants. Gene silencing is carried out by the CRISPR / Cas9 method, and it is aimed to prevent the role of the silenced gene in gene transcription. In this application, the permanent physiological effects of the targeted gene are not mentioned, and only its role in gene regulation is restricted. Since the gene in question regulates the transcription of multiple genes, different side effects can be expected as a result of gene silencing.

[0011] The patent application CN115011630A in the prior art relates to obtaining drought-resistant plants by overexpressing the SIAHL1 gene in tomato plants. With the plant mentioned in the said patent application, an increase in the number of living leaves and improvements in root length observed in reirrigation after drought were achieved, but no changes were observed in the stomatai structures involved in carbon dioxide and water uptake.

[0012] In the prior art [4], AHP2, AHP3 and AHP5 genes of Arabidopsis Thaliana were found to play a negative control role in the plant's response to drought stress. In the study mentioned above, when AHP2, AHP3 and AHP5 genes were interfered, and loss of function was realised, a drought-resistant phenotype was observed in the plant. The role of these genes in agricultural plants was not mentioned in the study, and the drought resistance effect was not associated with a decrease in stomatai number.

[0013] Previous techniques have targeted genes that negatively affect tomato plants' drought resistance and achieved silencing of these targeted genes by genetic mutation techniques. The fact that these genes targeted for tolerance in the technique are involved in more than one pathway in the organism increases the side effects of these mutations and may cause undesirable results.

[0014] The limitations and inadequacies of current state-of-the-art solutions, the fact that the genes targeted for drought resistance are active in multiple pathways, and the fact that the drought-resistant plants obtained do not contain critical drought protective physiological effects, such as reduced stomatai number, necessitated the development of a drought-resistant tomato plant. Brief Description and Objective of the Invention

[0015] The invention describes a genetically modified tomato plant in which the genes Solyc01g080540 with the nucleotide sequence SEQ ID NO: 9 and Solyc06084410 with the nucleotide sequence SEQ ID NO: 12 are mutated by the CRISPR / CAS9 method so that these genes are not expressed and thus acquire drought resistant properties, the method of obtaining this tomato plant and the gRNAs to be used in this method. Within the scope of the invention, gRNAs specifically designed to keep the target mutation rate above 80% are identified and cloned into the pHSE401 vector to ensure their introduction into the organism.

[0016] The invention aims to provide drought-resistant genetically modified tomato plants. For this purpose, the genes Solyc01g080540 with SEQ ID NO: 9 nucleotide sequence and Solyc06084410 with SEQ ID NO: 12 nucleotide sequence are mutated and silenced, and the expression of these genes is prevented, and a tomato plant with reduced stomata and developed root system is obtained. With the decrease in the number of stomata, the water loss of the plant in question decreases, and with the development of the root system, the plant's access to water becomes easier. Thanks to these features, the plant can better manage its water needs and increase its resistance to drought.

[0017] Another aim of the invention is to improve the plant's drought resistance by enhancing properties that prevent water loss while not interrupting photosynthesis. In this context, the number of stomata is reduced in the invention instead of closing the stomatai aperture that allows CO2 and water passage. Unlike the alternative of closing the stomatai aperture, reducing the number of stomata does not interrupt CO2 exchange in the inventive plant and does not adversely affect the continuity of photosynthesis.

[0018] Description of Figures

[0019] Figure 1 : Possible secondary structures of gRNAs targeting AHP genes with gRNA scaffold A) gRNA 3, B) gRNA 25, C) gRNA 29, D) gRNA 78 Figure 2: A) plant length graph, B) leaf water capacity (RWC) graph, C) basal angle graph, D) leaf temperature graph; in WT, ahp-10 and ahp-11 mutant plants compared to control and drought conditions

[0020] Figure 3: A) root length graph, B) lateral root number graph; in WT, ahp-10 and ahp-11 mutant plants compared under control and drought conditions

[0021] Figure 4: A) scanning electron microscopy (SEM) images of stomata, B) stomatai aperture graph, C) stomatai length graph, D) number of stomata per unit area graph, E) stomatai width graph; in WT, ahp-10 and ahp-11 mutant plants compared under control and drought conditions

[0022] Detailed Description of the Invention

[0023] The invention relates to a tomato plant with an improved root system and reduced stomatai number phenotype obtained by targeting and mutating Solyc01g080540 gene with SEQ ID NO: 9 sequence and Solyc06084410 gene with SEQ ID NO: 12 sequence by CRISPR / Cas9 gene editing method, which play a role in cytokinin signalling to increase drought resistance in tomato plants, the method by which this plant is obtained and the guide RNAs (gRNAs) designed to be used in this method.

[0024] The genes targeted in the invention are from the AHP gene family, and the protein sequence with sequence SEQ ID NO 7 encoded by the Solyc01g080540 gene with sequence SEQ ID NO 9 and the protein sequence with sequence SEQ ID NO 15 encoded by the Solyc06g084410 gene with sequence SEQ ID NO 12 are 70% similar to the AHP1 protein in Arabidopsis thaliana with 151 alignment length and 63% similar with 118 alignment length, respectively. However, these genes may show different functional properties in different organisms. Therefore, silencing mutations in different AHP proteins are not exactly expected to show drought resistance effect in tomato plants.

[0025] The AHP protein, the product of the AHP gene used in the invention, only acts as a signal transducer by transferring phosphate to Type-B and Type-A ARR proteins responsible for cytokinin signalling. Due to this limited property of the target gene, fewer unexpected intracellular effects are observed when the target gene is silenced compared to, for example, the inhibition of a transcription factor protein. In this way, the tomato plants obtained in the invention acquire drought resistance without showing undesirable side properties.

[0026] The production method of a drought-resistant genetically modified tomato according to the invention comprises the following process steps: i. Targeted silencing of SEQ ID NO:9 nucleotide sequence Solyc01g080540 and SEQ ID NO:12 nucleotide sequence Solyc06084410 genes with guide RNAs (gRNA) ii. Cloning of guide RNAs with Bsal cutting enzyme into a vector suitable for Agrobacterium-mediated transformation iii. Transfer of the cloned vector into Agrobacterium tumefaciens transformant iv. Cloning of Agrobacterium tumefaciens transformant into tomato plant v. Obtaining genetically modified tomato plants by tissue culture-mediated organogenesis process

[0027] In one implementation of the invention, gRNA 3 with sequence sequence SEQ ID NO: 1 , gRNA 25 with sequence SEQ ID NO: 2, gRNA 29 with sequence SEQ ID NO: 3 and gRNA 78 with sequence SEQ ID NO: 4 showing a working percentage of over 80% are used to mutate the target genes Solyc01g080540 with nucleotide sequence SEQ ID NO: 9 and Solyc06084410 with sequence SEQ ID NO: 12. These gRNAs can form stable secondary structures with a stem-loop arrangement as shown in Figure 1. These gRNAs were oligolated in accordance with the golden gate cloning method, primers in Table 1 were obtained, and the synthesised oligos were cloned into the pHSE401 vector with the nucleotide sequence of SEQ ID NO: 26. After cloning, the pHSE401 vector with sequence SEQ ID NO: 5 is transferred to Agrobacterium tumafaciens GV3101 and transferred to tomato plant cotyledons. Subsequently, mutant plants are obtained by organogenesis through tissue culture.

[0028] Table 1. Primers containing oligolated gRNA

[0029] The obtained ahp-10 and ahp-11 mutant tomato plant lines were grown in greenhouse conditions for 3 months in order to obtain seeds, and the T1 seeds harvested at the end of this period were sown under suitable conditions, and then the stress and analysis procedures were started.

[0030] At the end of 10 days of drought stress, wild-type (WT) plants lost their turgor and their growth was hampered. As can be seen in Figure 2. A, in terms of plant height, wild-type (WT) plants showed good growth under control conditions, while mutant plant lines (ahp-10 and ahp-11 ) showed better adaptation under drought conditions and were able to maintain their growth properly. Figure 2.B shows that in terms of leaf water capacity (Leaf RWC), mutant plants have a higher ratio compared to WT plants.

[0031] The roots of the plants exposed to drought stress were extracted from the soil, and their root development was analysed. Figure 3 shows the results of these analyses. It can be seen from the figure that mutant plants formed more lateral roots and longer main roots compared to WT plants, especially under drought. These features allow mutant plants to access water more easily than wild plants under drought conditions.

[0032] Changes in the stomatai structure of mutant plants at the end of drought stress compared to WT plants were also examined. SEM images and graphs of stomatai aperture, stomatai length, number of stomata per unit area and stomatai width are presented in Figure 4. As it can be seen from Figure 4, it is observed that regardless of the stress (i.e. under both control and drought conditions), fewer stomata are formed per unit area. Moreover, under drought stress, stomatai aperture was found to be less in mutant plants. With the decrease in the number of stomata and closure of the stomatai aperture, mutant plants can show drought resistance by experiencing less water loss under arid environmental conditions.

[0033] In one application of the invention, the Solyc01g080540 gene mutated in the ahp-10 tomato line obtained by transferring the vector with the sequence SEQ ID NO: 5 to the target plant has nucleotide sequence SEQ ID NO: 1 , and the protein resulting from the expression of this gene has the amino acid sequence SEQ ID NO: 8.

[0034] In another application of the invention, the Solyc06g084410 gene mutated in the ahp-10 tomato line obtained by transferring the vector with the sequence SEQ ID NO: 5 to the target plant has the nucleotide sequence SEQ ID NO:

[0035] 13 and the protein resulting from the expression of this gene has the amino acid sequence SEQ ID NO: 16.

[0036] In another application of the invention, the Solyc06g084410 gene mutated in the ahp-11 tomato line obtained by transferring the vector with the sequence SEQ ID NO: 5 to the target plant has the nucleotide sequence SEQ ID NO:

[0037] 14 and the protein resulting from the expression of this gene has the amino acid sequence SEQ ID NO: 17.

[0038] In another application of the invention, the Solyc01g080540 gene mutated in the ahp-11 tomato line obtained by transferring the vector with the sequence SEQ ID NO: 5 to the target plant has the nucleotide sequence SEQ ID NO: 11 and the protein resulting from the expression of this gene has the amino acid sequence SEQ ID NO: 6. REFERENCES

[0039] [1] Yan, l / V., Zhong, Y., & Shangguan, Z. (2017). Contrasting responses of leaf stomatai characteristics to climate change: a considerable challenge to predict carbon and water cycles. Global Change Biology, 23(9), 3781-3793.

[0040] [2] Han, S. K, Kwak, J. M., & Qi, X. (2021). Stomatai lineage control by developmental program and environmental cues. Frontiers in Plant Science, 12, 751852.

[0041] [3] Cui, Yingbo & Xu, Jiaming & Cheng, Minxia & Liao, Xiang-Ke & Peng,

[0042] Shaoliang. (2018). Review of CRISPR / Cas9 sgRNA Design Tools. Interdisciplinary Sciences: Computational Life Sciences. 10. 10. 1007 / sl 2539-018-0298-z.

[0043] [4] Nishiyama, R., Watanabe, Y., Leyva-Gonzalez, M. A., Van Ha, C., Fujita,

[0044] Y., Tanaka, M., Seki, M., Yamaguchi-Shinozaki, K, Shinozaki, K, & Herrera- Estrella, L. (2013). Arabidopsis AHP2, AHP3, and AHP5 histidine phosphotransfer proteins function as redundant negative regulators of drought stress response. Proceedings of the National Academy of Sciences, 110(12), 4840-4845

Claims

CLAIMS1. It is a drought-tolerant genetically modified tomato characterised by the absence of expression of the genes Solyc01g080540 with nucleotide sequence SEQ ID NO:9 and Solyc06084410 with nucleotide sequence SEQ ID NO:12.

2. A drought-tolerant genetically modified tomato according to claim 1 , characterised in that the mutated Solyc01g080540 gene has the nucleotide sequence SEQ ID NO:10 and the mutated Solyc06084410 gene has the nucleotide sequence SEQ ID NO:13.

3. A drought-tolerant genetically modified tomato according to claim 1 , characterised in that the mutated Solyc01g080540 gene has the nucleotide sequence SEQ ID NO:11 and the mutated Solyc06084410 gene has the nucleotide sequence SEQ ID NO:14.

4. A drought-tolerant genetically modified tomato according to claim 1 -3, characterised in that said tomato is Solanum lycopersicum.

5. It is a drought-tolerant genetically modified tomato production method characterised in that it includes the following process steps: i. Targeted silencing of SEQ ID NO:9 nucleotide sequence Solyc01g080540 and SEQ ID NO:12 nucleotide sequence Solyc06084410 genes with guide RNAs (gRNA) ii. Cloning of guide RNAs with Bsal cutting enzyme into a vector suitable for Agrobacterium-mediated transformation iii. Transfer of the cloned vector into Agrobacterium tumefaciens transformant iv. Cloning of Agrobacterium tumefaciens transformant into tomato plant v. Obtaining genetically modified tomato plants by tissue culture- mediated organogenesis process6. A method according to claim 5, characterised in that said guide RNAs are gRNA 3 with nucleotide sequence SEQ ID NO:1 , gRNA 25 with nucleotide sequence SEQ ID NO:2, gRNA 29 with nucleotide sequence SEQ ID NO:3 and gRNA 78 with nucleotide sequence SEQ ID NO:4.

7. A method according to claims 5 or 6, characterised in that said tomato is Solanum lycopersicum.

8. A method according to claims 5, 6 or 7, characterised in that said guide RNAs are cloned with the Bsal cutting enzyme into the vector pHSE401 with nucleotide sequence SEQ ID NO: 26.

9. A method according to any one of claims 5-8, characterised in that the transformant used is Agrobacterium tumafaciens GV3101 .

10. A drought-tolerant genetically modified tomato produced by a method according to any one of claims 5-9.

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