Tomato plants with improved whitefly resistance

Abstract

The present invention relates to tomato plants having improved insect resistance, more specifically whitefly or mite resistance, comprising the ASAT3 gene encoding an acetyl-CoA-dependent acyltransferase enzyme and the AP2e gene encoding the APETALA2 ethylene-responsive transcription factor. The present invention further relates to methods for preparing tomato plants with improved insect resistance and to the use of the ASAT3 gene in combination with the AP2e gene to provide insect-resistant tomato plants.

Description

[Technical Field]

[0001] The present invention relates to tomato plants having improved insect resistance, more specifically whitefly resistance, comprising the ASAT3 gene encoding an acetyl-CoA-dependent acyltransferase enzyme and the AP2e gene encoding the APETALA2 ethylene-responsive transcription factor. The present invention further relates to methods for preparing tomato plants with improved insect resistance and to the use of the ASAT3 gene in combination with the AP2e gene to provide insect-resistant tomato plants. [Background technology]

[0002] Economically, whiteflies are one of the most important pests of tomato crops. They cause direct damage by feeding on the plant's phloem sap and indirect damage by transmitting viruses and causing sooty diseases. Whitefly-related damage reduces the quality and quantity of crops, causing reduced plant vigor and yield, premature wilting, leaf chlorosis, and leaf drop.

[0003] Whiteflies belong to the family Aleyrodidae and typically feed on the abaxial side of plant leaves. Over 1,500 species have been described, and whiteflies are a major problem in crop protection in temperate and tropical climates, as well as in greenhouses, causing enormous economic losses worldwide each year. Many whitefly species are very small, making them difficult to control in greenhouses. If left unchecked, whitefly populations in greenhouses can quickly become overwhelming. Whitefly-related damage reduces the quality and quantity of crops, including reduced plant vigor and yield, premature wilting, leaf chlorosis, and defoliation. The silverleaf whitefly (Bemisia tabaci) is a species of whitefly that is currently one of the most important agricultural pests.

[0004] Although some species of whiteflies, when in very large numbers, can cause some crop losses simply by sucking sap, their primary damage is indirect. Their importance as crop pests lies in their role as vectors and the transmission of plant diseases. The primary problem posed by whiteflies is the transmission of viruses. Whiteflies are vectors of over 200 plant viruses, the most important of which belong to the Begomovirus genus (e.g., TYLCV) and various Criniviruses (e.g., ToCV) and Torrado viruses (ToANV, ToTV). Additionally, whiteflies feed by puncturing the plant's phloem, introducing toxic saliva and reducing the plant's overall turgor. Whiteflies secrete large amounts of honeydew, which promotes the infection of harmful fungi such as sooty mildew. Their large populations can quickly overwhelm susceptible plants.

[0005] Insecticides such as neonicotinoids, organochlorines, and organophosphate compounds are widely used and are effective methods for controlling whiteflies. However, repeated application of insecticides can lead to the development of resistance in whiteflies. Furthermore, more environmentally friendly biological methods have been proposed, such as using natural predators or parasitoids (e.g., lacewing larvae) to control whitefly infestations or washing plants to reduce the number of pests on the plants. However, these methods are not optimal solutions to the pests, and controlling whiteflies remains difficult.

[0006] Whitefly infestations are particularly problematic for tomatoes (Solanum lycopersicum) and peppers (Capsicum spp.). Tomatoes are classified in the Lycopersicon section of the Solanum genus, which contains 13 species. While the tomato is cultivated, the other 12 are wild relatives. The Capsicum genus contains 25 species, of which only five are cultivated: C. annuum, C. chinense, C. baccatum, C. pubescens, and C. frutescens. Domestication of tomatoes and peppers has resulted in a loss of genetic diversity and increased susceptibility to abiotic and biotic stresses, including pest attack. To date, no whitefly-resistant tomatoes or peppers have been cultivated. Several studies have been conducted to identify whitefly resistance in wild relatives of tomatoes and peppers. Some wild relatives of tomato (S. pennellii, S. habrochaites, S. peruvianum, and S. pimpinellifolium) are known to be more resistant than cultivated species. Resistance to whitefly may be due to higher levels of virus resistance and greater durability of the virus resistance genes used, due to lower selection pressure on virus resistance genes.

[0007] Antibiosis is one of the resistance mechanisms by which plants adversely affect the growth and survival of insects. One of the most prominent features of tomato that contributes to whitefly resistance is the trichome, a glandular hair-like structure or appendage of the plant. Their function is to secrete plant metabolic products, including terpenoids, phenylpropanoids, flavonoids, methyl ketones, and acyl sugars, which have diverse functions in plant growth and development and stress responses. For example, mono- and sesquiterpenes, methyl ketones, and acyl sugars are secondary metabolites known to be associated with whitefly resistance in tomato. While glandular trichomes appear to play an important role in whitefly resistance, the compounds within the trichomes are crucial. A high correlation has been found between the presence of specific trichomes (type IV trichomes) and whitefly resistance. Previous studies have shown that whitefly resistance is based on several mechanisms involving multiple genes, resulting in a complex trait. Efforts to introduce whitefly resistance into cultivated tomato have been unsuccessful, and new approaches and sources of resistance need to be explored. Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the above, there is a need in the art for tomato plants with improved insect resistance. Additionally, there is a need in the art for methods for providing plants with improved insect resistance, more particularly tomato plants with improved resistance to whiteflies.

[0009] It is an object of the present invention, inter alia, to address the above-mentioned needs in the art. This object of the present invention is achieved, inter alia, by the invention as outlined in the appended claims. [Means for solving the problem]

[0010] Specifically, the above objects are achieved, inter alia, according to a first aspect, by the present invention providing a tomato plant with improved whitefly resistance, the tomato plant comprising a combination of an acetyl-CoA-dependent acyltransferase gene (ASAT3) encoding a cDNA sequence having at least 95%, preferably at least 98%, more preferably at least 99% sequence identity with SEQ ID NO: 1, and an APETALA2e ethylene-responsive transcription factor gene (AP2e) encoding a cDNA sequence having at least 95%, preferably at least 98%, more preferably at least 99% sequence identity with SEQ ID NO: 3, wherein said combination of ASAT3 and AP2e genes improves C phenotype in tomato plants compared to tomato plants not comprising said combination of genes. 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , C 40 H 70 O 15 This is achieved by a tomato plant that results in an increase in the acyl sugar content of one or more sugars selected from the group consisting of, preferably all of said sugars. More preferably, the ASAT3 gene encodes the cDNA of SEQ ID NO: 2, and the AP2e gene encodes the cDNA of SEQ ID NO: 4. Experiments have shown that the introgression and combination of the ASAT3 and AP2e genes into commercial tomatoes contributes to the production of specific acylsucroses, which have a distinct effect on whitefly toxicity, resulting in increased whitefly resistance in tomato plants. Correlation analysis between individual acylsucroses and whitefly mortality revealed that differences in individual acylsucrose molecules play different roles in the resistance of tomato plants to whiteflies. Comparison of whitefly mortality with individual acylsucroses revealed that a specific acylsucrose compound, C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C40 H 70 O 15 showed a correlation coefficient of 0.5 or more, indicating a high impact on resistance. 。 Bioassay experiments in tomato plants according to the present invention show that at the identified acyl sugar concentrations of the specifically indicated acyl sugars, a level of resistance in whitefly mortality, i.e., an increase in WF resistance, of at least 50%, preferably at least 55%, more preferably at least 60%, and even more preferably at least 65% is observed. Plants that do not contain the ASAT3 and / or AP2e genes in their genome do not exhibit the identified acyl sugar concentrations of the specifically indicated acyl sugars and this level of resistance.

[0011] The tomato plants of the present invention, preferably tomato plants, have improved insect resistance, and the plants contain an ASAT3 gene in combination with an AP2e gene. The APETALA2 (AP2) gene family (sometimes referred to as the AP2 / ethylene-responsive element-binding factor (ERF) gene family or ERF / AP2 gene family) defines a large gene family (>100+ genes) of DNA-binding proteins called AP2 / ERF in tomato plants. AP2 genes perform various functions, including hormone regulation, establishment of floral meristem organ identity, regulation, growth, and development of floral organs, as well as various responses to environmental stimuli and stress responses. Furthermore, it is known that various different AP2 genes cause changes in the ratio of hexose to sucrose during plant seed development, and that AP2 proteins regulate the amount of sugars in the system and are involved in the transport, shaping, and signaling in the plant using these various sugars. Surprisingly, the ASAT3 gene in combination with the AP2e gene binds to C, a specific S4-type acyl sugar. 39 H 66 O 15 (S4: dC27 acylsucrose), C 39 H 68 O 15 (S4:C27 acylsucrose), C 38 H 66 O 15(S4: C26 acylsucrose) and C 40 H 70 O 15 It has been found that ASAT3 promotes and regulates the production of S4:C28 acylsucroses, and specifically increased production of these acylsugars is associated with high levels of insect resistance in plants. The ASAT3 gene encodes an acyl-CoA-dependent acyltransferase (type III) enzyme that can acylate the furanose ring of diacylsucroses. ASAT3 appears to be involved in the final step of the acylsugar pathway, catalyzing the acylation of triacylsucroses to produce specific tetraacylsucroses.

[0012] The AP2e gene encodes the APETALA2e ethylene-responsive transcription factor, which is involved in regulating acyl sugar production. While AP2e is involved in the ability to produce general amounts of different types of acyl sugars, ASAT3 appears to promote the production of specific S4 acyl sugars, which affect plant insect resistance. While AP2e influences the total amount of acylsucrose produced by switching on genes involved in the biosynthetic pathway and trichome formation, ASAT3 appears to catalyze or affect the final step in the acyl sugar pathway, more specifically the step that converts S3 to S4 acyl sugars. The active ASAT3 enzyme appears to be responsible for adding an additional acyl group to acylsucroses that already contain three acyl groups. This results in increased amounts of specific tetraacylsucroses, leading to improved insect resistance, as observed in the tomato plants of the present invention. When the ASAT3 gene was absent in tomato plants, the plants showed reduced resistance to whiteflies compared to tomato plants containing the ASAT3 gene.

[0013] Experiments show that the type of acyl sugar is crucial for conferring whitefly resistance to tomato plants. It was observed that plants containing the AP2e gene and producing acyl sugars were not necessarily resistant to whiteflies. However, when plants also contained the ASAT3 gene, the plants were able to tolerate certain tetraacyl (S4) sucrose, more specifically C, compared to susceptible plants. 39 H66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15 The present invention shows improved insect resistance associated with increased levels of

[0014] Compared to plants that do not contain the ASAT3 gene in combination with the AP2e gene, the tomato plants of the present invention have increased S4 acyl sugar content. Acyl sugars are found in trichome exudates, and more specifically, C 39 H 66 O 15 (S4: dC27 acylsucrose), C 39 H 68 O 15 (S4:C27 acylsucrose), C 38 H 66 O 15 (S4: C26 acylsucrose), and C 40 H 70 O 15 Acyl sugars (S4:C28 acylsucrose) are Type IV trichome exudates and confer insect resistance in tomato plants. AP2e is involved in both trichome development and acyl sugar production. Tomato plants containing the AP2e gene exhibit increased Type IV trichomes on the leaf surface and stems. Most tomato trichome exudates (approximately 90%) contain acyl sugars, of which over 70 compounds have been identified. The acyl sugars produced in tomato consist of various combinations of acyl groups, derived from aliphatic acids of various chain lengths esterified to the hydroxyl groups of glucose or sucrose. The acyl chains are primarily short- to medium-length aliphatic acids, either branched or linear. In tomato, the major short acyl chains of acyl sugars have been shown to be derived from acetic acid (C2) or branched-chain amino acids, namely 2-methyl-propanoic acid (C4) and 3-methyl-butanoic acid (C5). Longer acyl groups are likely derived from beta-oxidation products of fatty acids. However, the presence and abundance of specific acyl sugars differ significantly between resistant and susceptible plants, especially C39 H 66 O 15 (S4: dC27 acylsucrose), C 39 H 68 O 15 (S4:C27 acylsucrose), C 38 H 66 O 15 (S4: C26 acylsucrose), and C 40 H 70 O 15 (S4:C28 acylsucrose) The acyl sugar content is high in insect-resistant plants. These acyl sugars are sticky substances that act as adhesive traps and are also toxic to insects, more specifically whiteflies, improving the insect resistance of the plant. Furthermore, whiteflies as well as other pierce-sucking insects avoid settling on leaves in the presence of these specific acyl sugars.

[0015] According to a preferred embodiment, the present invention relates to a tomato plant, wherein the whitefly is one or more selected from the group consisting of Aleurocanthus woglumi (citrus spiny whitefly), Aleyrodes proletella (bean whitefly), Bemisia tabaci (silverleaf whitefly), Trialeurodes vaporariorum (greenhouse whitefly), preferably T. vaporariorum and / or B. tabaci.

[0016] According to a preferred embodiment of the present invention, the plant of the present invention as detailed above is not a plant obtained essentially exclusively by biological processes.

[0017] The genomic regions or fragments of the present invention can be introduced into tomato plants by introgression. However, since the nucleotide sequences of the genomic fragments of the present invention are known, these genomic fragments can be artificially constructed in yeast and then recombined with a susceptible tomato genome. Alternatively, these genomic regions or fragments can be amplified by long-range PCR amplification, and the resulting amplified fragments can be transformed into plant cells in a single step, or by a series of transformations to ultimately obtain tomato plants of the present invention. The genomic fragments of the present invention can also be completely or partially reconstituted later, for example, isolated from a gel or column after restriction digestion, and then transformed into tomato cells. Furthermore, mutations, deletions, or insertions in the genome can be obtained by EMS mutagenesis and / or CRISPR technology. Alternatively, the genomic fragment of interest can be introduced into a vector under a (strong) promoter. Resistance can then be obtained by transforming a susceptible plant with the vector and expressing the desired sequence. These techniques are readily available to those skilled in the art. The construction of artificial chromosomes containing the genomic fragments of the present invention is also contemplated within the context of the present invention.

[0018] According to yet another preferred embodiment, the present invention relates to a tomato plant, wherein the ASAT3 gene encodes the protein sequence represented by SEQ ID NO:2 and the AP2e gene encodes the protein sequence represented by SEQ ID NO:4.

[0019] According to yet another preferred embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15The present invention relates to tomato plants having a combined acyl sugar content of at least 200 μg / g of fresh weight (FW) of plant leaves, preferably at least 250 μg / g of fresh weight (FW) of plant leaves, and more preferably at least 300 μg / g of fresh weight (FW) of plant leaves. Fresh weight (FW) refers to the mass of a plant or plant part, in this case, plant leaves, at harvest. Bioassay experiments in tomato plants according to the present invention show that at identified acyl sugar concentrations of the specifically indicated acyl sugars, resistance levels of whitefly mortality of at least 50%, preferably at least 55%, more preferably at least 60%, and even more preferably at least 65% are observed.

[0020] According to another preferred embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: 39 H 66 O 15 The tomato plant has an acyl sugar content of at least 1 μg / g of FW of the plant leaves, preferably at least 1.5 μg / g of FW of the plant leaves, and more preferably at least 2 μg / g of FW of the plant leaves. 39 H 66 O 15 Although present in small amounts in view of the other identified acyl sugars in tomato plants showing increased WF resistance, this particular acyl sugar appears to be the one that most likely causes or contributes most to the toxic effect on WF and contributes to the plant's resistance.

[0021] According to a preferred embodiment, the present invention provides a 39 H 68 O 15 The present invention relates to a tomato plant having an acyl sugar content of at least 200 μg / g of FW of plant leaves, preferably at least 250 μg / g of FW of plant leaves, more preferably at least 300 μg / g of FW of plant leaves.

[0022] According to another preferred embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: 38 H 66 O 15The present invention relates to a tomato plant having an acyl sugar content of at least 5 μg / g of FW of plant leaves, preferably at least 10 μg / g of FW of plant leaves, more preferably at least 15 μg / g of FW of plant leaves.

[0023] According to yet another preferred embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: 40 H 70 O 15 The present invention relates to a tomato plant having an acyl sugar content of at least 5 μg / g of FW of plant leaves, preferably at least 10 μg / g of FW of plant leaves, more preferably at least 15 μg / g of FW of plant leaves.

[0024] According to yet another preferred embodiment, the present invention relates to a tomato plant obtainable from deposit NCIMB 44054. Most preferably, the present invention relates to a tomato plant in which the AP2e gene and the ASAT3 gene are obtainable from, derived from, or originating from the tomato plant deposited on October 14, 2022 with NCIMB Ltd, Aberdeen, Scotland under number NCIMB 44054.

[0025] According to another preferred embodiment, the present invention relates to tomato plants that are additionally resistant to mites, preferably spider mites (Tetranychus urticae).

[0026] According to another preferred embodiment, the present invention relates to a tomato plant, wherein the acetyl-CoA-dependent acyltransferase gene (ASAT3) is present in the genome of the plant in at least a heterozygous, preferably a homozygous, form.

[0027] According to another preferred embodiment, the present invention relates to a tomato plant in which the APETALA2e ethylene-responsive transcription factor gene (AP2e) is present in the plant genome at least in a heterozygous state, preferably in a homozygous state. Experiments have shown that whitefly resistance appears to be optimal when the plant genome contains both AP2e and ASAT3 in a homozygous state. However, plants in which the ASAT3 gene is heterozygous also exhibit whitefly resistance.

[0028] According to a preferred embodiment, the present invention relates to a tomato plant, wherein said plant is a tomato.

[0029] According to another preferred embodiment, the present invention relates to a tomato plant, wherein said plant does not contain in its genome the SlAT2 gene encoding the cDNA sequence of SEQ ID NO:5.

[0030] According to yet another preferred embodiment, the present invention provides a tomato plant comprising a combination of ASAT3 and AP2e genes, which is characterized by a C phenotype of tomato plant compared to a tomato plant not comprising said combination of genes. 32 H 54 O 15 When the tomato plant contains the AP2e and ASAT3 genes but does not contain the SlAT2 gene, in addition to the four identified sugars, the tomato plant further increases the acyl sugar content of C 32 H 54 O 15 also likely contributes to the increased WF resistance phenotype of the tomato plants of the present invention.

[0031] According to a second aspect, the present invention relates to seeds, fruits or plant parts of the tomato plants of the invention.

[0032] According to a further aspect, the present invention provides a method for providing a tomato plant with improved whitefly resistance, the method comprising the steps of providing a whitefly-susceptible tomato plant; - providing a combination of an acetyl-CoA-dependent acyltransferase gene (ASAT3) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:1 and an APETALA2e ethylene-responsive transcription factor gene (AP2e) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:3, wherein said combination of ASAT3 and AP2e genes increases C compared to tomato plants not comprising said combination of genes. 39 H 66 O 15 (S4: dC27 acylsucrose), C 39 H 68 O 15 (S4:C27 acylsucrose), C 38 H 66 O 15 (S4:C26 acylsucrose), C 40 H 70 O 15 (S4: C28 acylsucrose) and C 32 H 54 O 15 mutating its genome to result in an increased acyl sugar content (S4:C20 acylsucrose); The present invention relates to a method comprising:

[0033] According to a further aspect, the present invention provides a method for preparing a tomato plant with improved whitefly resistance, comprising the steps of: a) crossing a whitefly susceptible tomato plant with the tomato plant disclosed above; b) selecting tomato plants with improved insect resistance, which contain the AP2e gene and the ASAT3 gene; The present invention relates to a method comprising:

[0034] According to a preferred embodiment, the present invention relates to a method for providing a tomato plant with improved whitefly resistance, wherein the presence of the AP2e gene in said tomato plant with improved insect resistance is determined using the markers SEQ ID NO: 7 and SEQ ID NO: 8, and the presence of the ASAT3 gene is determined using the markers SEQ ID NO: 9 and SEQ ID NO: 10. Furthermore, the selection of whitefly-resistant tomato plants can also be performed by determining or identifying the specific sequences (cDNA or protein sequences) of ASAT3 and AP2e, identified herein as SEQ ID NOs: 1, 2, 3 and 4, respectively.

[0035] According to another preferred embodiment, the present invention provides a method for providing tomato plants with improved whitefly resistance, wherein the selection of tomato plants with improved insect resistance comprises the step of: 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 and / or C. 40 H 70 O 15 By determining the acyl sugar content, C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 and C 40 H 70 O 15 The combined acyl sugar content of the above is at least 200 μg / g of fresh weight (FW) of plant leaves, and / or C 39 H 66 O 15 and / or C 39 H 68 O 15 and / or C 38 H 66 O15 Apart from determining the presence of AP2e and ASAT3 genes in tomato plants, the selection of tomato plants with improved insect resistance can be carried out by using C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15 This can be done based on determining the acyl sugar content.

[0036] According to a further aspect, the present invention provides a method for preparing a tomato plant with improved whitefly resistance, comprising the steps of: a) providing a tomato plant as described herein, comprising an AP2e and an ASAT3 gene; b) crossing the tomato plant of step a) with a tomato plant that is more susceptible to whiteflies and does not contain the AP2e and ASAT3 genes; c) optionally self-pollinating the plants obtained in step b) at least once; d) selecting plants with improved whitefly resistance; The present invention relates to a method comprising:

[0037] According to a further aspect, the present invention relates to a combination of an acetyl-CoA-dependent acyltransferase gene (ASAT3) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:1 and an APETALA2e ethylene-responsive transcription factor gene (AP2e) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:3 for providing insect resistance to tomato plants.

[0038] According to a further aspect, the present invention relates to the use of a combination of the two genes AP2 and ASAT3 as defined above in tomato plants to provide whitefly-resistant tomato plants.

[0039] The invention is explained in more detail in the following examples and figures. [Brief explanation of the drawings]

[0040] [Figure 1] 1 shows the leaves of a tomato plant (Lycopersicon esculentum) according to the invention (top) and an insect-susceptible tomato plant (Lycopersicon esculentum) (bottom). Both tomato plants were exposed to whitefly infestation in vitro in glass Petri dishes. The leaves of the plant according to the invention are free of viable whitefly infestation, with most of the whiteflies being deformed and dead. In contrast, the leaves of the insect-susceptible tomato plant are clearly infested with whiteflies, with live, healthy whiteflies. [Figure 2] This figure shows the percentage of whitefly (WF) mortality in response to increasing concentrations of acyl sugars on tomato plants. The graph shows a correlation between whitefly mortality and the concentrations of the acyl sugars C39H66O15, C39H68O15, C38H66O15, and C40H70O15. These specific acyl sugars are found to negatively affect whitefly survival. In contrast, other acyl sugars present in the plant, such as C34H58O15, do not affect whiteflies. Fresh mass (FW) refers to the mass of the plant, in this case the FW of the plant leaves at the time of harvest. This bioassay demonstrates that the level of acyl sugars present in the plant leaves directly influences the level of resistance, as observed by whitefly mortality. [Figure 3]Figure 3A shows the average toxicity level of whiteflies (WF) determined in tomato plants containing homozygotes (+ / +) or heterozygotes (+ / -) for the ASAT3 gene, or in the absence (- / -) of the ASAT3 gene, as well as the average amounts of C39H66O15, C39H68O15, C38H66O15, and C40H70O15 acyl sugars (shown as S4-type acyl sugars in μg / g Fw in Figure 3B and in plants). The absence / presence of the ASAT3 gene is directly related to the toxicity level of WF and the amount of these specific sugars in tomato plants. All plants contained homozygotes for the AP2e gene. A plant is considered WF-resistant if the observed WF toxicity is greater than 50%, more preferably greater than 60%. [Figure 4] This figure shows the expression of the AP2e and ASAT3 genes determined by RT PCR using β-actin as an endogenous control. The expression value of each gene was normalized to the endogenous β-actin gene. The expression levels of the AP2e and ASAT3 genes were measured in whitefly-resistant and susceptible tomato plants. Three types of plants were included: plant A homozygous for the AP2e gene (+ / +) and heterozygous for the ASAT3 gene (+ / -), plant B heterozygous for both AP2e and ASAT3, and plant C deleted for AP2e (- / -) and heterozygous for ASAT3. The presence / absence of the AP2e gene correlates with the expression of the ASAT3 gene and the WF toxicity level and the amount of specific sugars in tomato plants. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0041] Excised leaf bioassay Young leaf segments from the third leaf were excised from tomato plants at least 12 weeks old. The petioles of the leaf segments were then placed in test tubes filled with nutrient agar to prevent dehydration. The test tubes were then placed (using Blu-Tack) in the center of glass Petri dish lids, with ample space between the leaf and the Petri dish, with the adaxial side facing up and the abaxial side facing down. Each Petri dish was inoculated with 25 whiteflies and anesthetized with CO2 for 3 seconds.

[0042] After 24-48 hours, the number of whiteflies on the adaxial or abaxial surface was counted, along with the number of dead whiteflies. Each plant was tested twice, and the percentage of dead whiteflies was calculated by comparing the number of surviving whiteflies (those feeding on the adaxial and abaxial portions of the leaves and those still flying in the dish). Correlation analysis between specific acylsucroses and whitefly mortality revealed that differences in specific acylsucrose molecules play different roles in resistance / susceptibility to whiteflies.

[0043] As shown in Figure 2, which compares the mortality rate of whiteflies (WF) with the specific acylsucrose content in the plant body, mainly C 39 H 66 O 15 (S4: dC27 acylsucrose), C 39 H 68 O 15 (S4:C27 acylsucrose), C 38 H 66 O 15 (S4: C26 acylsucrose), and C 40 H 70 O 15 The acyl sugars (S4:C28 acylsucrose) have a significant effect on resistance. For example, C 37 H 64 O 15 (S4:C25), C 32 H 56 O 14 (S3:C20), C 34 H 58 O 15No such significant relationship was observed between insect-susceptible and -resistant plants for the acyl sugar content of (S4:C22).

[0044] Analysis of acyl sugar content in tomatoes by liquid chromatography-mass spectrometry (LC-MS) Chemical analysis of leaf surfaces by LC-MS was performed on a series of tomato plants, including insect-resistant, intermediate-resistant, and susceptible plants (all tomatoes) according to the present invention. Plants were grown to 10 axillary buds, and leaf pieces were collected from the third leaf by placing a cap and opening on both sides of the leaf piece's surface and pressing a 15 ml test tube (1.5 cm diameter) once. The perforated leaf pieces were then immersed in 2 ml of methanol plus octaacetylsucrose (10 mg / L) and shaken for 15 seconds. The perforated leaf pieces were then removed and 600 μl transferred to a 0.5 ml 96-well plate. The methanol was evaporated, and the samples were redissolved in 300 μL of methanol and analyzed using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6230 TOF mass spectrometer.

[0045] A 1 μL extract was injected and separated on an Agilent ZORBAX RRHD Eclipse Plus C18 column at 50 °C with a mobile phase flow rate of 0.3 mL / min. The mobile phase consisted of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B). The A:B gradient was from 60:40 to 45:55 in 6 min, to 10:90 in 8 min, and to 60:40 in 3 min. Molecules were ionized at 325 eV (positive mode) and detected in the 50–1500 μm range at 1 spectrum / second. The extract primarily contained acyl sugars, which were detected as sodium adducts by mass spectrometry.

[0046] Individual acyl sugars were identified by calculating molecular formulas based on the sodium and potassium adducts that comprised the chromatographic peaks using MassHunter Qualitative Analysis software (Agilent), where the molecular formulas were calculated based on the carbon, hydrogen, and oxygen atoms in the adducts. + , Na + , K.+ The adduct ions were paired with α- and β-glucose, and the double bond equivalent (DBE) range was further constrained to 1–10. The combination of the accurate mass of the adduct ions and the DBE allowed us to estimate the basic structure of the acyl sugar molecule, including the backbone, the number of acyl chains, and the total number of carbon atoms forming the acyl chain. The amount of acyl sugar was calculated by integrating the peaks in the chromatogram using MassHunter Quantitative Analysis Software (Agilent) and comparing the total peak area of ​​each individual acyl sugar with that of the internal standard (octaacetylsucrose).

[0047] LC-MS also produced results comparable to those obtained in the excised leaf bioassays described above, indicating that specific acyl sugars are involved in insect resistance. The S4-type acyl sugar compounds shown above are present in high concentrations in plants that exhibited high resistance to whiteflies. In contrast, these specific acyl sugars were not detected or were detected in low concentrations by LC-MS in plants that were susceptible to whiteflies. The LC-MS results indicated that plants with improved insect resistance were characterized by C 39 H 66 O 15 (S4: dC27 acylsucrose), C 39 H 68 O 15 (S4:C27 acylsucrose), C 38 H 66 O 15 (S4: C26 acylsucrose), and C 40 H 70 O 15 It can be concluded that this is related to the high acyl sugar content of (S4:C28 acylsucrose). 37 H 64 O 15 (S4:C25), C 32 H 56 O 14 (S3:C20), C 34 H 58 O 15 No significant changes in the acyl sugar content of (S4:C22) were observed in the insect susceptibility and resistance of plants.

[0048] Genotyping, mapping of ASAT3 and AP2e The production of acyl sugars in tomato plants is associated with a high level of insect resistance in the plants. It is important to know which type of acyl sugar is required for insect resistance. Examining the genotype data of a resistant tomato plant population (Lycopersicon esculentum) by marker analysis, it was found that the C 39 H 66 O 15 (S4: dC27 acylsucrose), C 39 H 68 O 15 (S4:C27 acylsucrose), C 38 H 66 O 15 (S4: C26 acylsucrose), and C 40 H 70 O 15 (S4:C28 acylsucrose) A QTL positively correlated with acylsucrose production was identified.

[0049] Briefly, the genomic region associated with the amount of acyl sugars produced by type IV trichomes was mapped on chromosome 6 based on the reference genome SL2.40. The region involved in acyl sugar production associated with insect resistance was determined to be located between positions 43250794 bp and 43259933 bp. Marker M5 is 100% associated with the amount of acyl sugars produced. Based on the reference genome SL2.40 and in silico prediction analysis (ITAG 2.3), one gene, Solyc06g075510.2, is located within the fine-mapped region, encoding the APETALA2 ethylene-responsive transcription factor (AP2e).

[0050] Furthermore, the type of acyl sugar is crucial for conferring whitefly resistance to tomato plants. It has been observed that plants containing the AP2e gene and producing acyl sugars do not necessarily exhibit resistance to whiteflies. Comparing the acyl sugar profiles of susceptible and resistant plants, plants with improved insect resistance have higher levels of tetraacyl (S4) sucrose C compared to susceptible plants that are unable to produce the above series of S4 sugars or can only produce triacyl sucrose (S3). 39 H 66 O 15 (S4: dC27 acylsucrose), C 39 H 68 O 15 (S4:C27 acylsucrose), C 38 H 66 O 15 (S4: C26 acylsucrose), and C 40 H 70 O 15 It was concluded that the marker M1 is 100% associated with the type of acyl sugar and maps to a specific sequence on chromosome 11, which encodes a member of the BAHD family of acyltransferases, more specifically the acetyl-CoA-dependent acyltransferase enzyme ASAT3, which can acetylate acylsugars and C 39 H 66 O 15 (S4: dC27 acylsucrose), C 39 H 68 O 15 (S4:C27 acylsucrose), C 38 H 66 O 15 (S4: C26 acylsucrose), and C 40 H 70 O 15 It is involved in the production of (S4:C28 acylsucrose).

[0051] Determination of the nucleotide sequence of the functional ASAT3 gene resulted in the identification of a genomic sequence including the promoter region. Plants containing functional ASAT3 in combination with AP2e (SEQ ID NO: 1) have increased acyl sugar content and are more resistant to whiteflies than plants not containing SEQ ID NO: 1. SEQ ID NO: 1 shows the coding sequence of ASAT3 in the plant of the present invention, which encodes the ASAT3 protein (SEQ ID NO: 2). SEQ ID NO: 3 shows the coding sequence of AP2e in the plant of the present invention, which encodes the AP2e protein (SEQ ID NO: 4).

[0052] [Table 1]

[0053] The ASAT3 gene in combination with the AP2e gene encodes a specific type of S4 acyl sugar, C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15 The combination of AP2e (marker M5) + ASAT3 (marker M1) increases the level of specific S4 acylsucrose required for whitefly resistance. Using the M5 and M1 markers, several tomato plants were selected for the presence of the AP2e gene and the absence or presence of the ASAT3 gene, homozygous or heterozygous. The total acyl sugar content per plant (μg per gram plant fresh weight (gFW)) was determined, as well as the specific acyl sugar C 39 H 66 O 15、 C 39 H 68 O 15、 C 38 H 66 O 15 and C 40 H 70 O 15 The presence of ASAT3 and resistance to whiteflies were determined.39 H 66 O 15、 C 39 H 68 O 15、 C 38 H 66 O 15 and C 40 H 70 O 15 It co-segregates with acylsucrose production and is associated with whitefly resistance levels, see Table 2.

[0054] [Table 2]

[0055] Furthermore, in additional experiments, the whitefly toxicity level and C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15 The average amount of acyl sugars was determined as described above in plants containing homozygotes (+ / +) or heterozygotes (+ / -) for the ASAT3 gene, or in plants where the ASAT3 gene was absent (- / -). Figure 3A shows that the relative whitefly (WF) toxicity in plants containing the ASAT3 gene was superior to that in plants lacking ASAT3, with homozygotes providing the best WF resistance. Figure 3B shows that the C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15The average sum of acyl sugars (designated as S4-type acyl sugars) is directly related to the presence or absence of the ASAT3 gene, indicating that homozygous plants produce the highest average amount of that particular sugar. As long as the ASAT3 gene is present in combination with the AP2e gene, the plants are more resistant to whiteflies and C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15 It appears to be more productive with acyl sugars.

[0056] AP2e and ASAT3 gene expression levels and WF resistance in tomato The expression levels of the AP2e and ASAT3 genes were measured in whitefly-resistant and susceptible tomato plants. Three types of plants were included: plant A, homozygous for the AP2e gene (+ / +) and heterozygous for the ASAT3 gene (+ / -), plant B, heterozygous for both AP2e and ASAT3, and plant C, deleted for AP2e (- / -) and heterozygous for ASAT3. Furthermore, specific S4 acyl sugar C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15 The acyl sugar content of AP2e and ASAT3 was measured and whitefly resistance was determined as described above. See Table 3 for a summary of the results per plant. AP2e homozygous and ASAT3 heterozygous plants had a high acyl sugar content of C. 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C40 H 70 O 15 Plants heterozygous for both genes produce the most C. 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15 The amount of acyl sugars produced is low, and therefore the toxicity to WF is low.

[0057] [Table 3]

[0058] RNA was extracted from the plants. Briefly, small pieces of stem from each plant were placed in 50 ml test tubes and frozen in liquid nitrogen. The tubes were then vortexed to remove the trichomes from the samples, which were then allowed to settle to the bottom of the tubes. The tubes were then returned to liquid nitrogen. RNA extraction was performed using Quiagen's RNeasy Plant Mini Kit according to the manufacturer's instructions. RNA content was measured using Quiagen's nanodrop.

[0059] Next, 1 μg of RNA was treated with DNase I (Thermo Scientific) according to the manufacturer's instructions. The RNA was reverse transcribed using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit according to the manufacturer's instructions. For the reverse transcription reaction, total RNA was split into two independent reactions: 500 ng was used in a reaction containing reverse transcriptase, and the other 500 ng was used in a reaction without enzyme as a negative control. The resulting cDNA was diluted 3-fold by adding 40 μl of nuclease-free water.

[0060] One μl of cDNA was loaded into a qPCR reaction with 10 μl of Promega Go-Taq qPCR Master Mix, 0.4 μl of each primer (Table 4) at 10 μM, and nuclease-free water to a final volume of 20 μl. Relative standard curves were also generated for each primer pair by performing two-fold serial dilutions starting with the original three-fold diluted cDNA. Expression analysis was performed using the ΔΔCt method, with β-actin as the endogenous control. Expression values ​​for each gene were normalized to the endogenous β-actin gene (Figure 4).

[0061] [Table 4]

Claims

1. 1. A tomato plant having improved whitefly resistance, comprising a combination of an acetyl-CoA-dependent acyltransferase gene (ASAT3) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:1, and an APETALA2e ethylene-responsive transcription factor gene (AP2e) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:3, wherein the combination of ASAT3 and AP2e genes increases C > 100% compared to a tomato plant not comprising the combination of genes. 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , C 40 H 70 O 15 1. A tomato plant, wherein the tomato plant has an increased acyl sugar content of one or more sugars selected from the group consisting of:

2. The tomato plant of claim 1, wherein the ASAT3 gene encodes the protein sequence represented by SEQ ID NO: 2 and the AP2e gene encodes the protein sequence represented by SEQ ID NO:

4.

3. C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , and C 40 H 70 O 15 3. The tomato plant according to claim 1, wherein the combined acyl sugar content of the above is at least 200 μg / g of fresh mass (FW) of plant leaves, preferably at least 250 μg / g of fresh mass (FW) of plant leaves, and more preferably at least 300 μg / g of fresh mass (FW) of plant leaves.

4. C 39 H 66 O 15 4. The tomato plant according to claim 1, wherein the acyl sugar content of the tomato plant is at least 1 μg / g of FW of the plant leaves, preferably at least 1.5 μg / g of FW of the plant leaves, and more preferably at least 2 μg / g of FW of the plant leaves.

5. C 39 H 68 O 15 5. The tomato plant according to claim 1, wherein the acyl sugar content of the tomato plant is at least 200 μg / g of FW of the plant leaves, preferably at least 250 μg / g of FW of the plant leaves, more preferably at least 300 μg / g of FW of the plant leaves.

6. C 38 H 66 O 15 6. The tomato plant according to claim 1, wherein the acyl sugar content of the tomato plant is at least 5 μg / g of FW of the plant leaves, preferably at least 10 μg / g of FW of the plant leaves, more preferably at least 15 μg / g of FW of the plant leaves.

7. C 40 H 70 O 15 7. The tomato plant according to claim 1, wherein the acyl sugar content of the tomato plant is at least 5 μg / g of FW of the plant leaves, preferably at least 10 μg / g of FW of the plant leaves, more preferably at least 15 μg / g of FW of the plant leaves.

8. 8. The tomato plant of any one of claims 1 to 7, which is available from deposit NCIMB 44054.

9. 9. The tomato plant according to any one of claims 1 to 8, which is additionally resistant to mites, preferably spider mites (Tetranychus urticae).

10. 10. The tomato plant according to claim 1, wherein the ASAT3 gene is present in the genome of the plant in at least a heterozygous, preferably a homozygous, form.

11. 11. The tomato plant according to claim 1, wherein the AP2e gene is present in the genome of the plant in at least a heterozygous, preferably a homozygous, form.

12. 12. The tomato plant of claim 1, which is a tomato (Solanum lycopersicum var. cerasiforme).

13. 13. The tomato plant of claim 1, wherein the tomato plant does not contain in its genome the SlAT2 gene encoding the cDNA sequence of SEQ ID NO:

5.

14. The combination of ASAT3 and AP2e genes increases C compared to tomato plants that do not contain said combination of genes. 32 H 54 O 15 14. The tomato plant of claim 1, further resulting in an increased acyl sugar content of

15. 15. A seed, fruit or plant part of a tomato plant according to any one of claims 1 to 14.

16. 15. A method for providing a tomato plant with improved whitefly resistance according to any one of claims 1 to 14, comprising the steps of providing a whitefly-susceptible tomato plant; - providing a combination of an acetyl-CoA-dependent acyltransferase gene (ASAT3) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:1 and an APETALA2e ethylene-responsive transcription factor gene (AP2e) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:3, wherein said combination of ASAT3 and AP2e genes increases C compared to tomato plants not comprising said combination of genes. 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 , C 40 H 70 O 15 mutating its genome, comprising increasing the acyl sugar content of one or more of: A method comprising:

17. 1. A method for selecting tomato plants with improved whitefly resistance, comprising: a) crossing a whitefly susceptible tomato plant with a tomato plant according to any one of claims 1 to 14, b) selecting S. lycopersicum plants with improved insect resistance, which contain the AP2e gene and the ASAT3 gene; A method comprising:

18. 18. The method of claim 17, wherein the presence of the AP2e gene in the S. lycopersicum plants with improved insect resistance is determined using markers SEQ ID NO:7 and SEQ ID NO:8, and the presence of the ASAT3 gene is determined using markers SEQ ID NO:9 and SEQ ID NO:

10.

19. Selection of S. lycopersicum plants with improved insect resistance was performed in C. 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 and / or C. 40 H 70 O 15 By determining the acyl sugar content, C 39 H 66 O 15 , C 39 H 68 O 15 , C 38 H 66 O 15 and C 40 H 70 O 15 The combined acyl sugar content of the above is at least 200 μg / g of fresh weight (FW) of plant leaves, and / or C 39 H 66 O 15 and / or C 39 H 68 O 15 and / or C 38 H 66 O 15 The method according to claim 17 or 18, wherein the acyl sugar content of the plant leaf extract is at least 5 μg / g relative to the FW of the plant leaf.

20. 1. A method for preparing a tomato plant with improved whitefly resistance, comprising: a) providing a tomato plant according to any one of claims 1 to 14, comprising AP2e and ASAT3 genes; b) crossing the tomato plant of step a) with a tomato plant that is more susceptible to whiteflies and does not contain the AP2e and ASAT3 genes; c) optionally self-pollinating the plants obtained in step b) at least once; d) selecting plants with improved whitefly resistance; A method comprising:

21. A combination of an acetyl-CoA-dependent acyltransferase gene (ASAT3) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:1 and an APETALA2e ethylene-responsive transcription factor gene (AP2e) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:3 for providing insect resistance to tomato plants.

22. 22. Use of the combination of the ASAT3 gene and AP2e gene according to claim 21 in a tomato plant to provide a whitefly-resistant tomato plant.

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