Tomato plant having improved insect resistance

Abstract

To provide a tomato plant with improved insect resistance, particularly resistance to whiteflies or mites, the tomato plant including an SlAT2 gene that encodes an acetyl-CoA-dependent acyltransferase enzyme and an AP2e gene that encodes an APETALA2 ethylene-responsive transcription factor.SOLUTION: The present invention relates to methods for preparing a tomato plant having improved insect resistance, and to use of an SlAT2 gene in combination with an AP2e gene for providing an insect-resistant tomato plant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to tomato plants having improved insect resistance, more particularly whitefly or mite resistance, comprising the SlAT2 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 SlAT2 gene in combination with the AP2e gene to provide insect-resistant tomato plants. [Background technology]

[0002] 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.

[0003] 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 comes from their role as vectors and the transmission of plant diseases, including over 200 plant viruses. 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. Because whiteflies aggregate in large numbers, susceptible plants can quickly become overwhelmed.

[0004] 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.

[0005] 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, S. pimpinellifolium) are known to be more resistant than cultivated species.

[0006] 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. Trichomes 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, it is the compounds within the trichome that are crucial. A high correlation has been found between the presence of specific trichomes (type IV trichomes) and whitefly resistance, and previous studies have demonstrated that whitefly resistance is a complex process based on several mechanisms involving multiple genes. 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]

[0007] In view of the above, there is a need in the art for tomato plants with improved insect resistance, more particularly 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 for tomato plants with improved resistance to whiteflies.

[0008] 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]

[0009] Specifically, the above objects are achieved, inter alia, according to a first aspect, the present invention provides a tomato plant with improved whitefly resistance, the tomato plant comprising a combination of an acetyl-CoA-dependent acyltransferase gene (SlAT2) encoding a cDNA sequence having at least 95%, preferably at least 98%, more preferably at least 99% sequence identity with SEQ ID NO:2, 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:5, wherein said combination of SlAT2 and AP2e genes improves C phenotype in tomato plants compared to tomato plants not comprising said combination of genes. 29 H 48 O 15 and C 36 H 62 O 15 This is achieved by increasing the acyl sugar content in tomato plants. More preferably, the SlAT2 gene encodes the cDNA of SEQ ID NO: 2, and the AP2e gene encodes the cDNA of SEQ ID NO: 5.

[0010] The tomato plants of the present invention, preferably tomato plants, have improved insect resistance, and the plants contain an SlAT2 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 SlAT2 gene in combination with the AP2e gene binds to a specific acyl sugar, C29 H 48 O 15 (S4: C17 acylsucrose) and / or C 36 H 62 O 15 It has been found that the promotion and regulation of the production of S4:C24 acylsucroses, as well as the specific increased production of these acylsugars, are associated with high levels of insect resistance in plants. The SlAT2 gene encodes an acetyl-CoA-dependent acyltransferase enzyme, and the AP2e gene encodes the APETALA2e ethylene-responsive transcription factor, which is involved in regulating acylsugar production. While AP2e is responsible for the ability to produce general amounts of different types of acylsugars, SlAT2 appears to promote the production of specific acylsugars that 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, SlAT2 has a strong influence on the type of acylsucrose ultimately produced. The active SlAT2 enzyme is responsible for adding an additional acetyl group to acylsucroses that already contain three acyl groups. This results in increased amounts of tetraacylsucrose at the expense of triacylsucrose, improving insect resistance as observed in tomato plants. This is an additional step in the chain of reactions catalyzed by a different SlAT-type enzyme than SlAT2, a different acyl-Cotransferase that only adds up to three acyl chains to the initial sucrose molecule and therefore only indirectly contributes to insect resistance.

[0011] According to another preferred embodiment, the present invention relates to a tomato plant comprising tetraacyl (S4) sugars and triacyl (S3) sugars, wherein the ratio of tetraacyl (S4) sugars to triacyl (S3) sugars (S4:S3) in the plant is at least 1, preferably at least 1.2, more preferably at least 1.5, and most preferably at least 1.7. Experiments have shown that 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. However, when the plant also carries the SlAT2 gene, the plant exhibits improved insect resistance, which is due to the presence of high levels of tetraacyl (S4) sucrose, e.g., high C, compared to susceptible plants that accumulate mostly triacyl sucrose (S3). 29 H 48 O 15 (S4:C17) and C 36 H 62 O 15 (S4:C24).

[0012] Compared to plants that do not contain the SlAT2 gene in combination with the AP2e gene, the tomato plants of the present invention have 29 H 48 O 15 and / or C. 36 H 62 O 15 The acyl sugar content is increased. Acyl sugars are found in trichome exudates, more specifically, C 29 H 48 O 15 (S4: C17 acylsucrose) and C 36 H 62 O 15Acyl sugars (S4:C24 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 C 29 H 48 O 15 and C 36 H 62 O 15 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.

[0013] 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.

[0014] 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.

[0015] 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 spinach 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.

[0016] According to another preferred embodiment, the present invention relates to a tomato plant, wherein the genomic region encoding the SlAT2 gene has at least 95%, preferably at least 98%, more preferably at least 99% sequence identity with SEQ ID NO: 1, and the genomic region encoding the AP2e gene has at least 95%, preferably at least 98%, more preferably at least 99% sequence identity with SEQ ID NO: 4. SEQ ID NO: 1 and SEQ ID NO: 4 are genomic regions containing the SlAT2 and AP2e genes, respectively, including their respective promoter elements.

[0017] According to yet another preferred embodiment, the present invention relates to a tomato plant, wherein the SlAT2 gene encodes the protein sequence represented by SEQ ID NO:3 and the AP2e gene encodes the protein sequence represented by SEQ ID NO:6.

[0018] According to another preferred embodiment, the present invention relates to a tomato plant, wherein the SlAT2 gene encodes the coding sequence of SEQ ID NO:2 and the AP2e gene encodes the coding sequence of SEQ ID NO:5.

[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: 29 H 48 O 15 and / or C, wherein the acyl sugar content of the plant leaf is at least 150 μg / g of fresh mass (FW) of the plant leaf, preferably at least 200 μg / g of FW of the plant leaf, more preferably at least 250 μg / g of FW of the plant leaf. 36 H 62 O 15 The present invention relates to tomato plants having an acyl sugar content of at least 125 μg / g of FW of plant leaves, preferably at least 175 μg / g of FW of plant leaves, and more preferably at least 250 μg / g of FW of plant leaves. Fresh mass (FW) is the mass of the plant or plant part, in this case the plant leaves, at harvest. Bioassay experiments show that at the claimed acyl sugar concentrations, resistance levels of an average of 40% or more whitefly mortality are observed.

[0020] According to yet another preferred embodiment, the present invention relates to a tomato plant, available from deposit NCIMB 43748 of NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, Scotland, on March 18, 2021.

[0021] According to another preferred embodiment, the present invention provides a method for producing a C β -actin-containing plant, which is characterized by ... 28 H 46 O15 , C 34 H 58 O 15 and C 35 H 60 O 15 The present invention relates to a tomato plant further having an increased content of one or more acyl sugars selected from the group consisting of acyl sucrose compound C. 29 H 48 O 15 , C 36 H 62 O 15 , C 28 H 46 O 15 , C 34 H 58 O 15 , C 35 H 60 O 15 followed by two additional sugars C 33 H 56 O 15 and C 37 H 64 O 15 was found to be present in higher (although not significantly) concentrations in resistant tomato plants compared to their susceptible counterparts.

[0022] According to yet another preferred embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: 28 H 46 O 15 and / or C, wherein the acyl sugar content of the plant leaf is at least 10 μg / g of fresh mass (FW) of the plant leaf, preferably at least 15 μg / g of fresh mass (FW) of the plant leaf, more preferably at least 20 μg / g of fresh mass (FW) of the plant leaf, and / or C 34 H 58 O 15 and / or C, wherein the acyl sugar content of the plant leaf is at least 15 μg / g of fresh mass (FW) of the plant leaf, preferably at least 20 μg / g of fresh mass (FW) of the plant leaf, more preferably at least 25 μg / g of fresh mass (FW) of the plant leaf, and / or 35 H 60 O 15is at least 12.5 μg / g of fresh mass (FW) of plant leaves, preferably at least 15 μg / g of fresh mass (FW) of plant leaves, more preferably at least 20 μg / g of fresh mass (FW) of plant leaves.

[0023] According to another preferred embodiment, the present invention relates to tomato plants that are additionally resistant to mites, preferably spider mites (Tetranychus urticae).

[0024] According to a second aspect, the present invention relates to seeds, fruits or plant parts of the tomato plants of the invention.

[0025] 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 (SlAT2) encoding a cDNA sequence having at least 95% sequence identity with SEQ ID NO:2, and an APETALA2e ethylene-responsive transcription factor gene (AP2e) encoding a cDNA sequence having at least 95% sequence identity with SEQ ID NO:5, wherein said combination of SlAT2 and AP2e genes increases C compared to tomato plants not comprising said combination of genes. 29 H 48 O 15 and C 36 H 62 O 15 mutating its genome, including resulting in an increased acyl sugar content.

[0026] 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 a defined whitefly resistant tomato plant of the invention; b) selecting a tomato plant having improved insect resistance, the tomato plant comprising the SlAT2 gene and the AP2e gene. The selection of a tomato plant having improved insect resistance is carried out by 29 H 48 O 15 and / or C. 36 H 62 O 15 Determination of acyl sugar content can be performed based on C 29 H 48 O 15 and / or C, wherein the acyl sugar content of the plant leaf is at least 150 μg / g of fresh mass (FW) of the plant leaf, preferably at least 200 μg / g of FW of the plant leaf, more preferably at least 250 μg / g of FW of the plant leaf. 36 H 62 O 15 The acyl sugar content of the S1AT2 and AP2e is at least 125 μg / g of FW of the plant leaves, preferably at least 175 μg / g of FW of the plant leaves, more preferably at least 250 μg / g of FW of the plant leaves. Furthermore, the selection of whitefly-resistant tomato plants can also be achieved by determining or identifying the specific sequences (cDNA, gDNA or protein sequences) of S1AT2 and AP2e, identified herein as SEQ ID NO: 1 to SEQ ID NO: 6.

[0027] According to another preferred embodiment, the present invention provides that the selection of tomato plants with improved insect resistance is carried out by C. 29 H 48 O 15 and / or C. 36 H 62 O 15 By determining the acyl sugar content, C 29 H 48 O 15 The acyl sugar content of the plant leaf is at least 150 μg / g of fresh weight (FW), and / or C 36 H 62 O 15 is at least 125 μg / g of fresh weight (FW) of plant leaves.

[0028] According to a further aspect, the present invention relates to a combination of two genomic regions for providing insect resistance to tomato plants, wherein one genomic region comprises SEQ ID NO:1 encoding an acetyl-CoA-dependent acyltransferase gene (SlAT2) and a second genomic region comprises SEQ ID NO:4 encoding an APETALA2e ethylene-responsive transcription factor gene (AP2e).

[0029] According to a further aspect, the present invention relates to a combination of two genes for providing insect resistance to tomato plants, wherein one gene encodes an acetyl-CoA-dependent acyltransferase (SlAT2) protein comprising SEQ ID NO:3 and a second gene encodes an APETALA2e ethylene-responsive transcription factor (AP2e) comprising SEQ ID NO:6.

[0030] According to a further aspect, the present invention relates to the use of a combination of two genomic regions or two genes as defined above in tomato plants to provide whitefly resistant tomato plants.

[0031] The invention is explained in more detail in the following examples and figures. [Brief explanation of the drawings]

[0032] [Figure 1] 1 shows leaves (A and B) of a tomato plant (tomato) according to the invention and leaves (C and D) of an insect-susceptible tomato plant (tomato). Both tomato plants were exposed to whitefly infestation in a commercial greenhouse where whitefly infestation was promoted. The leaves of the plant according to the invention are free of whitefly infestation, whereas the leaves of the insect-susceptible tomato plant are clearly infected with whiteflies. FIG. E shows leaves of an insect-susceptible tomato plant (S) and a tomato plant according to the invention (R). It is clear that the whiteflies are alive and present on the surface of the leaves of the S plant, whereas the whiteflies are dead and absent from the surface of the R plant. [Figure 2A]Figure 2A shows the percentage of dead whiteflies (WF) on tomato plants as a function of increasing concentrations of acyl sugars. The graph in Figure 2A shows a linear relationship between the number of dead whiteflies and the concentration of the acyl sugar C29H48O15. [Figure 2B] Figure 2B shows the percentage mortality of whiteflies (WF) on tomato plants as a function of increasing acyl sugar concentrations. Graph 2B shows the dose response of C36H62O15 acyl sugars. Both of the specific acyl sugars negatively impact whitefly survival. [Figure 2C] This figure shows the percentage of whitefly (WF) mortality in response to increasing concentrations of acyl sugars on tomato plants. In contrast, other acyl sugars present in plants, such as C32H54O15 (graph 2C), do not themselves 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] Overlaid LC-MS chromatograms show the presence and relative concentrations of various acyl sugars in insect-resistant plants (red peak), intermediate-resistant plants (orange peak), and susceptible plants (green peak). Red-labeled acyl sugar compounds are present in higher concentrations in insect-resistant plants and are thought to play an important role in plant insect resistance. Green-labeled acyl sugars are primarily present in susceptible plants. From this analysis, it can be concluded that plants with improved insect resistance are associated with higher levels of C29H48O15 (S4:C17) and C36H62O15 (S4:C24) acyl sugars. Furthermore, no significant changes related to plant insect resistance were observed in the acyl sugar contents of C27H46O14 (S3:C15), C32H56O14 (S3:C20), C33H58O14 (S3:C21), C34H60O14 (S3:C22), and C39H68O15 (S4:C27). [Figure 4-1]FIG. 1 shows the genomic region (gDNA), coding sequence (cDNA) and protein sequence of SlAT2 (SEQ ID NOs: 1 to 3, respectively), and the gDNA, cDNA and protein sequences of AP2e (SEQ ID NOs: 4 to 6, respectively), and shows that SlAT2 in combination with AP2e confers whitefly resistance in tomato plants. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 4-3] This is a continuation of Figure 4-2. [Figure 4-4] This is a continuation of Figure 4-3. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0033] Excised leaf bioassay Young leaves approximately 4 cm long were excised from the tops of approximately 50 tomato plants, each at least 12 weeks old, grown in a greenhouse. The petioles were placed in test tubes containing nutrient agarose gel. The test tubes were placed horizontally (using Blu-Tack) against the wall of a glass Petri dish, with the adaxial side of the leaf facing up, at a medium height, leaving space between the leaf and the dish on both the adaxial and abaxial sides. Each dish was inoculated with 25 whiteflies and anesthetized with CO2 for 3 seconds.

[0034] 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 around in the petri dish) with the number of dead whiteflies. Correlation analysis between specific acylsucroses and whitefly mortality revealed that differences in specific acylsucrose molecules play different roles in resistance / susceptibility to whiteflies. As shown in Figure 2, which compares whitefly (WF) mortality and acylsucrose content in plants, the main role was C 29 H 48 O 15 (S4:C17) and C36 H 62 O 15 The acyl sugar (S4:C24) had a significant effect on resistance. Other acyl sugar compounds that were associated with resistance were C 28 H 46 O 15 (S4:C16), C 34 H 58 O 15 (S4:C22), C 35 H 60 O 15 (S4:C23), C 33 H 56 O 15 (S4:C21), and C 37 H 64 O 15 (S4:C25) is an acyl sugar.

[0035] 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 two opposing leaf segments (3 x 3 x 3 cm) from the third or fourth terminal leaf were placed in a 10 ml glass vial. Two ml of methanol containing an internal standard (octaacetylsucrose, 10 mg / L) was added, and the vial was shaken for 15 seconds. The leaf segments were removed, and 300 μl of the methanol extract was transferred to an LC vial and analyzed using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6230 TOF mass spectrometer.

[0036] 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.

[0037] Individual acyl sugars were identified by calculating their molecular formulas based on the parent ions that comprised the chromatographic peaks using MassHunter Qualitative Analysis software (Agilent). The molecular formulas were calculated based on the carbon, hydrogen, and oxygen atoms that formed the parent ions, and the H + , Na + , and K + The mass of the parent ion was combined with the double bond equivalent (DBE) to reveal the formic acid adduct, and the double bond equivalent (DBE) range was further constrained to 1–10. The combination of the accurate mass of the parent ion and the DBE allows estimation of the basic structure of the acyl sugar molecule, including the backbone moiety, 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 acyl sugar with that of the internal standard (octaacetylsucrose).

[0038] LC-MS yielded results comparable to those obtained in the excised leaf bioassays described above. Further evidence that specific acyl sugars are involved in insect resistance can be derived from the LC-MS chromatogram plot, Figure 3. Individual chromatograms of methanol leaf immersions of insect-resistant (red), intermediately resistant (orange), and susceptible (green) plants are overlaid. The acyl sugar compounds labeled in red are present in high concentrations in plants that exhibited high resistance to whiteflies. In contrast, these specific acyl sugars were either not detected or detected at low concentrations by LC-MS in plants that were susceptible to whiteflies. Furthermore, for whitefly-susceptible plants, the predominantly present acyl sugars were labeled in green, and notably, were absent or at low concentrations in resistant plants. This analysis suggests that plants with improved insect resistance are characterized by C 29 H 48 O 15 (S4:C17) and C 36 H 62 O 15 (S4:C24) It can be concluded that this is related to the abundance of acyl sugars. 27 H 46 O 14 (S3:C15), C 32 H 56 O 14 (S3:C20), C 33 H 58 O 14 (S3:C21), C 34 H 60 O 14 (S3:C22), and C 39 H 68 O 15 No significant changes in the acyl sugar content of (S4:C27) were observed in the insect susceptibility and resistance of plants.

[0039] Genotyping, mapping of SlAT2 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 genotypic data of a resistant tomato plant population (Lycopersicon esculentum) by marker analysis, it was found that the markers M8 and M5 (Table 1) were used to detect the acyl sugars (C 36 H 62 O 15 )(S4:C24) and (C 29 H 48 O 15 )(S4:C17) identified a QTL that was positively correlated with acylsucrose production.

[0040] 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 (Table 1). 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).

[0041] 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, which accumulate mostly triacyl sucrose (S3). 29 H 48 O 15 (S4:C17) and C 36 H 62 O 15It was concluded that the marker M8 is 100% associated with the type of acyl sugar (Table 1) and maps to a specific sequence on chromosome 1, which encodes a member of the BAHD family of acyltransferases, more specifically the acetyl-CoA-dependent acyltransferase enzyme SlAT2, which can acetylate acyl sucrose and C 29 H 48 O 15 (S4:C17) and C 36 H 62 O 15 It is involved in the production of (S4:C24).

[0042] The nucleotide sequence of the functional SlAT2 gene was determined, resulting in a genomic sequence (SEQ ID NO: 1) containing the promoter region. Plants containing SEQ ID NO: 1, i.e., functional SlAT2 in combination with AP2e, have an increased S4 / S3 ratio and are highly resistant to whiteflies compared to plants not containing SEQ ID NO: 1, and these plants are unable to produce S4 sugars, which cause susceptibility to whiteflies. SEQ ID NO: 1 shows the genomic sequence containing the SlAT2 gene containing the promoter region of a whitefly-resistant plant of the present invention. SEQ ID NO: 2 shows the coding sequence of SlAT2 of a plant of the present invention, which encodes the SlAT2 protein of SEQ ID NO: 3. SEQ ID NO: 4 shows the genomic sequence of the AP2e gene containing the promoter region of a whitefly-resistant plant of the present invention. SEQ ID NO: 5 shows the coding sequence of AP2e of a plant of the present invention, which encodes the AP2e protein of SEQ ID NO: 6.

[0043] [Table 1]

[0044] SlAT2 in combination with the AP2e gene 29 H 48 O 15 (S4: C17 acylsucrose) and / or C 36 H 62 O 15It was found that AP2e specifically promotes and regulates the production of certain types of acyl sugars, such as S4:C24 acylsucrose, and more generally increases the ratio of tetraacylated (S4) to triacylated (S3) sugars. The combination of AP2e (marker M5) and SlAT2 (marker M8) increases the levels of S4:C17 and S4:C24 acylsucrose required for whitefly resistance. Using the M5 and M8 markers, several tomato plants were selected for the presence of the AP2e gene and the absence or presence of the SlAT2 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 29 H 48 O 15 (S4: C17 acylsucrose) and C 36 H 62 O 15 The presence of tetraacylated (S4) and triacylated (S3) sucrose (S4:C24 acylsucrose), the ratio of tetraacylated (S4) to triacylated (S3) sucrose, and resistance to whiteflies were determined. The SlAT2 genotype cosegregated with the production of S4:C17 and S4:C24 acylsucrose and an increased S4 / S3 ratio, which correlated with the level of whitefly resistance (Table 2).

[0045] [Table 2]

Claims

1. 2. A tomato plant having improved whitefly resistance, comprising a combination of an acetyl-CoA-dependent acyltransferase gene (SlAT2) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:2, and an APETALA2e ethylene-responsive transcription factor gene (AP2e) encoding a cDNA sequence having at least 95% sequence identity to SEQ ID NO:5, wherein the combination of SlAT2 and AP2e genes exhibits improved whitefly resistance compared to a tomato plant not comprising the combination of genes. 29 H 48 O 15 and C 36 H 62 O 15 tomato plants, resulting in an increase in acyl sugar content.

2. 2. The tomato plant of claim 1, comprising tetraacyl (S4) sugars and triacyl (S3) sugars, wherein the ratio of tetraacyl (S4) sugars to triacyl (S3) sugars (S4:S3) in the plant is at least 1, preferably at least 1.2, more preferably at least 1.5, and most preferably at least 1.

7.

3. 3. The tomato plant of claim 1, wherein the genomic region encoding the SlAT2 gene comprises SEQ ID NO: 1 and the genomic region encoding the AP2e gene comprises SEQ ID NO:

4.

4. 4. The tomato plant of claim 1, wherein the SlAT2 gene encodes the protein sequence represented by SEQ ID NO: 3 and the AP2e gene encodes the protein sequence represented by SEQ ID NO:

6.

5. C 29 H 48 O 15 The acyl sugar content of the plant leaf is at least 150 μg / g of fresh weight (FW), and / or C 36 H 62 O 15 5. The tomato plant of claim 1 , wherein the acyl sugar content of the tomato plant is at least 125 μg / g of FW of the plant leaves.

6. Compared to tomato plants that do not contain the combination of genes, C 28 H 46 O 15 , C 34 H 58 O 15 and C 35 H 60 O 15 6. The tomato plant of claim 1, further comprising an increased content of one or more acyl sugars selected from the group consisting of:

7. C 28 H 46 O 15 and / or C 34 H 58 O 15 and / or C 35 H 60 O 15 7. The tomato plant of claim 1, wherein the acyl sugar content of the tomato plant is at least 12.5 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 43748.

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. A seed, fruit or plant part of a tomato plant according to any one of claims 1 to 9.

11. 10. A method for providing a tomato plant with improved whitefly resistance according to any one of claims 1 to 9, comprising the steps of providing a whitefly-susceptible tomato plant; A combination of an acetyl-CoA-dependent acyltransferase gene (SlAT2) encoding a cDNA sequence having at least 95% sequence identity with SEQ ID NO:2 and an APETALA2e ethylene-responsive transcription factor gene (AP2e) encoding a cDNA sequence having at least 95% sequence identity with SEQ ID NO:5 is provided, wherein the combination of SlAT2 and AP2e genes enhances C phenotypes compared to tomato plants not containing the combination of genes. 29 H 48 O 15 and C 36 H 62 O 15 mutating its genome to result in an increased acyl sugar content.

12. 1. A method for preparing a tomato plant with improved whitefly resistance, comprising: a) crossing a whitefly susceptible tomato plant with a tomato plant according to any one of claims 1 to 9; b) selecting tomato plants with improved insect resistance, which contain the SlAT2 gene and the AP2e gene; A method comprising:

13. Selection of tomato plants with improved insect resistance has been reported in C. 29 H 48 O 15 and / or C. 36 H 62 O 15 By determining the acyl sugar content, C 29 H 48 O 15 The acyl sugar content of the plant leaf is at least 150 μg / g of fresh weight (FW), and / or C 36 H 62 O 15 The method of claim 12, wherein the acyl sugar content of the plant leaf is at least 125 μg / g of fresh weight (FW) of the plant leaf.

14. A combination of two genomic regions for providing insect resistance to tomato plants, wherein one genomic region comprises SEQ ID NO:1 encoding an acetyl-CoA-dependent acyltransferase gene (SlAT2), and a second genomic region comprises SEQ ID NO:4 encoding an APETALA2e ethylene-responsive transcription factor gene (AP2e).

15. A combination of two genes for providing insect resistance to tomato plants, wherein one gene encodes an acetyl-CoA-dependent acyltransferase (SlAT2) protein comprising SEQ ID NO:3 and a second gene encodes an APETALA2e ethylene-responsive transcription factor (AP2e) comprising SEQ ID NO:

6.

16. 16. Use of a combination of two genomic regions or two genes according to claim 14 or 15 in a tomato plant to provide a whitefly resistant tomato plant.

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