Plant protection agents based on antifreeze proteins

Abstract

The present invention relates to antifreeze proteins, in particular antifreeze proteins for their use in plant protection.The present invention also relates to nucleic acids encoding antifreeze proteins, methods for producing antifreeze proteins, plants comprising antifreeze proteins or nucleic acids encoding them, compositions of at least one antifreeze protein.The present invention particularly relates to the use of antifreeze proteins or compositions comprising them as plant protection agents, and methods for protecting plants from pests and / or abiotic stress.

Description

[Technical field]

[0001] The present invention relates to anti-frost proteins, in particular anti-frost proteins for their use in plant protection. The present invention also relates to nucleic acids encoding the anti-frost proteins, methods for producing the anti-frost proteins, plants comprising the anti-frost proteins or the nucleic acids encoding them, compositions of at least one anti-frost protein. The present invention particularly relates to the use of the anti-frost proteins or compositions comprising them as plant protection agents and methods for protecting plants. [Background technology]

[0002] Abiotic stresses, such as frost or drought stress, are of major concern in the agricultural industry, as plants are not always able to adjust appropriately to cold, drought, salinity, heat, toxins, etc. Abiotic stress refers to the negative impact of non-living factors on living organisms. Abiotic stress factors are naturally occurring factors, such as intense sunlight, temperature, or wind, that can cause damage to living organisms. Although abiotic stress affects animals as well, plants are particularly dependent on environmental factors and cannot actively change their position, and are therefore particularly susceptible to abiotic stress. Abiotic stress is the most detrimental factor for crop growth and productivity worldwide.

[0003] For example, drought stress is one of the major causes of yield loss within the agricultural industry. Similarly, freezing also contributes significantly to yield loss, at least in areas that do not have temperatures above freezing throughout the year.

[0004] One important way that plants combat drought stress is by closing their stomata. The main hormone regulating stomatal opening and closing is abscisic acid. Another important factor in dealing with drought stress and in water uptake and export are aquaporins. Aquaporins are integral membrane proteins that constitute membrane channels that transport water and other essential solutes.

[0005] One mechanism that contributes to frost damage is the formation of water crystals that can disrupt plant cell structure and function. Specifically, freezing causes plant cells to shrink, forcing water into the intercellular spaces where it can freeze and form ice crystals. Membranes break down and become leaky. As temperatures rise and thawing begins, water is osmotically absorbed back into the cells. If this occurs quickly, the tissue is undamaged, but if thawing is slow, water is removed from the cells, they become dehydrated, and "frost burn" occurs. Freezing stress can be countered, for example, by overhead watering if sufficient water is available, or by overhead shielding of the plant to keep radiant heat near the plant. The plant itself also secretes antifreeze proteins (AFPs) to confer frost tolerance. Summary of the Invention

[0006] The present application surprisingly shows that some AFPs are also chitinases.

[0007] Chitin has the chemical formula (C8H 13 O5N) n It is a polymer of N-acetylglucosamine, a derivative of the sugar glucose, with a structure similar to that of cellulose. Long-chain polysaccharides are present in a variety of different organisms across different clades. For example, chitin is the main component of the cell walls of fungi, the exoskeletons of arthropods such as crustaceans and insects, the radula of mollusks, and the scales of fish. Chitin has a structure equivalent to that of cellulose.

[0008] The biological conversion of chitin polysaccharide to shorter oligomers requires hydrolases that contain a conserved chitin-binding domain and a chitin-specific active site. A large number of chitinolytic enzymes are produced by various bacteria and fungi for the degradation of chitin as an energy source. All of them are glycosyl hydrolases, but differ with respect to reaction mechanism, thermostability and product properties [Patil et al., Enzyme Microb. Technol., 2000. 26: p. 473-483]. Chitinolytic hydrolases can be classified according to their mode of action. Endochitinases (EC 3.2.1.14) randomly bind to chitin polysaccharide chains and hydrolyze internal glycosidic bonds resulting in various fragment sizes ranging from dimers to polymers. In contrast, exochitinases (EC 3.2.1.29) bind to the reducing or non-reducing ends of chitin and release monomeric or, to a lesser extent, dimeric GlcNAc units. These enzymes are necessary for the complete degradation of chitin. Finally, chitobiase (EC 3.2.1.29) cleaves the GlcNAc dimers, releasing GlcNAc monomers [Tews et al., Nat. Struct. Biol., 1996. 3: p. 638-648]. Other enzymes such as cellulases and lysozymes are also known to exhibit some hydrolytic activity directed against chitin but not specific for these substrates [Wu et al., J Food Sci Technol, 2012. 49(6): p. 695-703;Aiba, Carbohydr Res, 1994. 261: p. 297-306].

[0009] Common pests of plants include fungi, insects and mollusks. Pest infestations can result in reduced yields and contamination of produce with unwanted by-products.

[0010] Chemical pesticides currently commercially applied include substances from the groups of organochlorines, organophosphates, carbamates, pyrethroids, triazines and neonicotinoids for use as insecticides, herbicides, fungicides and rodenticides. These pesticides are not only used on agricultural areas, but also on non-agricultural public urban green areas, sports grounds, pet shampoos, building materials or the bottom of ships to eliminate or prevent the presence of undesirable species. Numerous adverse health effects have been associated with chemical pesticides, and while high occupational, intentional or accidental exposure can lead to hospitalization or death, these substances have been critically reviewed, as exposure can occur via skin contact, ingestion of contaminated consumer products or inhalation, whereby they can be metabolized, excreted and stored or accumulated in body fat [Nicolopoulou-Stamati et al., Front. Public Health, 2016, 4:148].

[0011] An ideal insecticide should not only be harmless to human health, but also be environmentally friendly and as effective and specific as possible for protecting plants from a given pest. Furthermore, an insecticide should ideally avoid the development of resistance in the pest. There remains a need for new products that meet these criteria.

[0012] Thus, the present application addresses protection from both abiotic and biotic stresses (such as from pests) using AFPs.

[0013] [Description of the Invention] The present invention aims to overcome the problems of current plant protection agents by providing an antifreeze protein-based approach to abiotic stress, particularly freezing and drought stress, and / or pest control. Specifically, the approach of the present invention relies on antifreeze proteins to protect plants from biotic and / or abiotic stress by inducing the activation of defense mechanisms in plants (i.e., by inducing plant immunity). Plants have immune systems that allow them to defend themselves against a wide range of pathogens and abiotic stresses. Some antifreeze proteins can also specifically degrade chitin, which is not produced in humans or other higher animals, and are therefore expected to pose no risk to human food intake or to other non-target organisms. Furthermore, antifreeze proteins are completely biodegradable and therefore environmentally friendly. Besides this, given that chitin is the main structural component in pests such as fungi or insects, as well as in the radula of mollusks, it is not expected that such pests will easily develop resistance to antifreeze proteins with chitinolytic activity.

[0014] The inventors have found that some antifreeze proteins activate the plant immune system, a mechanism that makes plants more resistant to abiotic stress or to certain pests, such as fungi or insects.

[0015] The present inventors have also found that some antifreeze proteins with chitinolytic activity can actually be used to protect plants from pests such as fungi and insects, and from abiotic stresses.

[0016] The present application shows for the first time how antifreeze proteins can be applied as plant protection agents to combat pest infestations, for example through application of antifreeze proteins to the surface of a plant or part thereof.

[0017] Moreover, the present invention makes an important contribution to the prior art plant protection agents, since the antifreeze proteins and their subsequent use as plant protection agents provide different advantages compared to established substances. Such advantages include the absence of safety hazards during handling or pathogenicity upon entry into the food chain. Moreover, when compared to established substances, much lower concentrations of the antifreeze proteins according to the present invention are required to achieve plant protection. This can lead to cost savings.

[0018] Finally, the inventors have found that the application of an antifreeze protein according to the invention can be combined with the application of a prior art plant protection agent, thereby further enhancing plant protection.

[0019] Thus, the present invention provides the following preferred embodiments: [1] A composition comprising at least one type of antifreeze protein comprising a first amino acid sequence that is at least 70%, for example 100%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2. [2] The composition described in [1], wherein the first amino acid sequence is at least 70%, for example 100%, identical to the amino acid sequence set forth in SEQ ID NO:1. [3] The composition described in [1] or [2], wherein the antifreeze protein comprises the amino acid sequence set forth in SEQ ID NO:1. [4] The composition described in [1], wherein the first amino acid sequence is at least 70%, for example 100%, identical to the amino acid sequence set forth in SEQ ID NO:2. [5] The composition described in [1] or [4], wherein the antifreeze protein comprises the amino acid sequence set forth in SEQ ID NO:2. [6] The composition described in any one of [1] to [5], wherein the antifreeze protein essentially consists of a first amino acid sequence that is at least 70%, for example 100%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2. [7] The composition described in any one of [1] to [6], wherein the antifreeze protein consists of a first amino acid sequence that is at least 70%, for example 100%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2. [8] The composition according to any one of [1] to [7], which is a plant protection agent. [9] The composition according to any one of [1] to [8], which is a liquid composition.

[10] The composition according to any one of [1] to [9], which is an aqueous composition.

[11] The composition according to any one of [1] to

[10] , wherein the antifreeze protein is contained in the composition at a concentration of 0.01 mg / L (w / v) to 100 mg / L (w / v).

[12] The composition according to any one of [1] to

[11] , wherein the antifreeze protein is contained in the composition at a concentration of 0.1 mg / L (w / v) to 70 mg / L (w / v).

[13] The composition according to any one of [1] to

[12] , wherein the antifreeze protein is contained in the composition at a concentration of 5 mg / L (w / v) to 30 mg / L (w / v).

[14] The composition according to any one of [1] to

[13] , which contains polyvinyl alcohol.

[15] The composition according to any one of [1] to

[14] , comprising polyvinyl alcohol at a concentration of 6% (v / v) to 10% (v / v), preferably at a concentration of about 8% (v / v).

[16] Use of a composition comprising at least one antifreeze protein as a plant protection agent.

[17] Use of the composition according to

[16] , wherein the composition is any one of [1] to

[15] .

[18] Use of the composition according to

[16] or

[17] as a plant protection agent against abiotic stress and / or pests.

[19] Use of the composition described in any one of

[16] to

[18] , wherein the plant protection agent is for abiotic stress, the abiotic stress being optionally freezing stress or drought stress.

[20] Use of the composition according to any one of

[16] to

[19] , wherein the plant protection agent is for abiotic stress, and the abiotic stress is freezing stress.

[21] Use of the composition described in any one of

[16] to

[20] , wherein the plant protection agent is for abiotic stress, and the abiotic stress is drought stress.

[22] Use of the composition according to any one of

[16] to

[18] , wherein the plant protection agent is directed against a harmful organism, the harmful organism being optionally a chitin-containing organism such as a fungus or an insect.

[23] The use of the composition according to any one of

[16] to

[18] and

[22] , wherein the plant protection agent is against a harmful organism, the harmful organism being optionally a fungus or an insect.

[24] The use according to any one of

[16] to

[18] and

[22] to

[23] , wherein the pest is a fungus.

[25] The use according to any one of

[22] to

[24] , wherein the fungus is a species of the genus Puccinia, Fusarium or Septoria.

[26] The use according to any one of

[22] to

[25] , wherein the fungus is a Fusarium or Septoria species.

[27] The use according to any one of

[22] to

[26] , wherein the fungus is a Fusarium species, preferably Fusarium culmorum.

[28] The use described in any one of

[22] to

[24] and

[25] , wherein the fungus is a species of the genus Puccinia.

[29] The use described in any one of

[22] to

[25] and

[28] , wherein the fungus is Puccinia triticina.

[30] The use according to any one of

[16] to

[29] , wherein the plant is an arable crop, a fruit-bearing plant or a vegetable.

[31] The use according to any one of

[16] to

[30] , wherein the plant is an arable crop such as wheat.

[32] The use according to any one of

[16] to

[31] , wherein the composition is applied to a plant or a part thereof.

[33] The use according to any one of

[16] to

[32] , wherein the composition is applied to a plant or a part thereof before the occurrence of abiotic stress and / or pests.

[34] The use according to any one of

[16] to

[33] , wherein the part of the plant is a leaf, a fruit or a seed.

[35] The use according to any one of

[16] to

[34] , wherein the part of the plant is a leaf.

[36] The use according to any one of

[16] to

[34] , wherein the part of the plant is a seed.

[37] The use according to

[36] , wherein the composition is applied to seeds before sowing.

[38] The use described in any one of

[16] to

[37] , wherein the composition induces plant immunity.

[39] The use according to any one of

[16] to

[38] , wherein one or more further plant protection agents are applied to the plant or a part thereof, and the further plant protection agent is not an antifreeze protein.

[40] A method for protecting a plant from abiotic stress and / or pests comprising application to the plant or a portion thereof of a composition comprising at least one antifreeze protein.

[41] The method for protecting a plant according to

[40] , wherein the composition is any one of [1] to

[15] .

[42] The method for protecting a plant according to

[40] or

[41] , wherein the composition is applied to a plant or a part thereof prior to emergence of abiotic stress and / or pests.

[43] The method for protecting a plant according to any one of

[40] to

[42] , wherein the part of the plant is a leaf, a fruit or a seed.

[44] The method for protecting a plant according to any one of

[40] to

[43] , wherein the part of the plant is a leaf.

[45] The method for protecting a plant according to any one of

[40] to

[43] , wherein the part of the plant is a seed.

[46] The method for protecting plants according to

[45] , wherein the composition is applied to seeds before sowing.

[47] The method for protecting a plant according to any one of

[40] to

[46] , wherein the plant or plant part is immersed in the composition.

[48] ​​The method for protecting a plant according to any one of

[40] to

[47] , wherein the composition is applied by spraying onto a surface of a plant or a part of a plant, such as a seed, e.g., a coated seed.

[49] The method according to any one of

[40] to

[48] , wherein the method is for protecting a plant from abiotic stress, and the abiotic stress is, optionally, freezing stress or drought stress.

[50] The method according to any one of

[40] to

[49] , wherein the method is for protecting a plant from abiotic stress, and the abiotic stress is optionally freezing stress.

[51] The method according to any one of

[40] to

[50] , wherein the method is for protecting a plant from abiotic stress, and the abiotic stress is optionally drought stress.

[52] The method according to any one of

[40] to

[48] , wherein the method is for protecting a plant from a pest, and the pest is optionally a fungus or an insect.

[53] The method according to any one of

[40] to

[48] and

[52] , wherein the pest is a chitin-containing organism such as a fungus or an insect.

[54] The method according to any one of

[40] to

[48] and

[52] to

[53] , wherein the pest is a fungus.

[55] The method according to any one of

[52] to

[54] , wherein the fungus is a species of the genus Puccinia, Fusarium or Septoria.

[56] The method according to any one of

[52] to

[55] , wherein the fungus is a Fusarium or Septoria species.

[57] The method according to any one of

[52] to

[56] , wherein the fungus is a Fusarium species, preferably Fusarium culmorum.

[58] The method according to any one of

[52] to

[55] , wherein the fungus is a species of the genus Puccinia.

[59] The method according to any one of

[52] to

[55] and

[58] , wherein the fungus is Puccinia triticina.

[60] The method according to any one of

[40] to

[59] , wherein the plant is an arable crop, a fruit-bearing plant or a vegetable.

[61] The method according to any one of

[40] to

[60] , wherein the plant is an arable crop such as wheat.

[62] The method according to any one of

[40] to

[61] , wherein the composition induces plant immunity.

[63] The method according to any one of

[40] to

[62] , further comprising application of one or more additional plant protection agents to the plant or a part thereof, wherein the additional plant protection agent is not an antifreeze protein. [Brief description of the drawings]

[0020] [Figure 1] Figure 1. Use of AFPs with chitinase activity as plant protection agents. F. curmorum growth and plant health were evaluated in the presence of AFPs with chitinase activity. (A) Control; (B) Control + F. curmorum; (C) Vaffr-2d + F. curmorum. [Diagram 2] Figure 1 shows the protective activity (damage reduction) of antifreeze proteins against freezing stress. Antifreeze protein Vaffr-2d was tested by foliar spray. Results are normalized to the control (CTL). [Diagram 3] Figure 1. Use of Vaffr-2d as a plant protection agent against abiotic stress. Pear yield after foliar application in the presence of freezing stress. [Figure 4] Figure 3. AFP-6 as an inducer of plant immunity. % Efficacy as a seed treatment against Puccinia triticina in Wheat Benchmark. [Diagram 5] Figure 3. AFP-6 as an inducer of plant immunity. (A) % efficacy of AFP-6 as a seed treatment against Puccinia triticina in Wheat Keitum. (B) % efficacy of Difend extra as a seed treatment against Puccinia triticina in Wheat Keitum. [Figure 6A] Figure 1. Effect of AFP-6 on the jasmonic and salicylic acid pathways. Seeds were treated with AFP-6 at 0.1 g / ton or 0.175 g / ton, and seedling samples were taken at 4, 7, 9, and 14 days post sowing (dps). (A) Fold change in LOX mRNA levels normalized to actin (upper panel) or ubiquitin (lower panel) is shown. [Figure 6B] (B) Effect of AFP-6 on the jasmonic and salicylic acid pathways. Seeds were treated with AFP-6 at 0.1 g / ton or 0.175 g / ton, and seedling samples were taken at 4, 7, 9, and 14 days post sowing (dps). (C) Fold change in OPR3 mRNA levels normalized to actin (upper panel) or ubiquitin (lower panel) is shown. [Figure 6C] Figure 1. Effect of AFP-6 on the jasmonic and salicylic acid pathways. Seeds were treated with AFP-6 at 0.1 g / ton or 0.175 g / ton, and seedling samples were taken at 4, 7, 9, and 14 days post sowing (dps). (C) Fold change in PR1-3 mRNA levels normalized to actin (upper panel) or ubiquitin (lower panel) is shown. [Figure 6D] Figure 1. Effect of AFP-6 on the jasmonic and salicylic acid pathways. Seeds were treated with AFP-6 at 0.1 g / ton or 0.175 g / ton, and seedling samples were taken at 4, 7, 9, and 14 days post sowing (dps). (D) Fold change in PR1-17 mRNA levels normalized to actin (upper panel) or ubiquitin (lower panel) is shown. [Figure 7A]Figure 1. Effect of Vaffr-2d on the jasmonic and salicylic acid pathways. Seeds were treated with Vaffr-2d at 0.175 g / ton and plants were sampled 6, 7, 8 and 11 days after sowing. (A) Fold change in OPR3 mRNA levels normalized to actin (upper panel) or ubiquitin (lower panel) is shown. [Figure 7B] (B) Effect of Vaffr-2d on the jasmonic and salicylic acid pathways. Seeds were treated with Vaffr-2d at 0.175 g / ton and plants were sampled 6, 7, 8 and 11 days after sowing. (C) Fold change in LOX mRNA levels normalized to actin (upper panel) or ubiquitin (lower panel) is shown. [Figure 7C] Figure 1. Effect of Vaffr-2d on the jasmonic and salicylic acid pathways. Seeds were treated with Vaffr-2d at 0.175 g / ton and plants were sampled 6, 7, 8 and 11 days after sowing. (C) Fold change in PR1-3 mRNA levels normalized to actin (upper panel) or ubiquitin (lower panel) is shown. [Figure 7D] Figure 1. Effect of Vaffr-2d on the jasmonic and salicylic acid pathways. Seeds were treated with Vaffr-2d at 0.175 g / ton and plants were sampled 6, 7, 8 and 11 days after sowing. (D) Fold change in PR1-17 mRNA levels normalized to actin (upper panel) or ubiquitin (lower panel) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of enzymology, plant protection, biochemistry, genetics, and molecular biology.

[0022] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0023] When used in the context of the present invention, the term "about" means that the value following the term "about" can vary within a range of ±20%, preferably within a range of ±15%, and more preferably within a range of ±10%.

[0024] All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. In case of conflict, the present specification, including definitions, will take precedence over the cited references. In addition, the materials, methods and examples are merely illustrative and are not intended to be limiting unless otherwise specified.

[0025] As used herein, each occurrence of a term such as "comprising" or "comprises" can be optionally replaced with "consisting" or "consisting". The term "consisting essentially of" in the context of a compound or composition means that there may be specific additional components present that do not substantially affect the essential properties of the compound or composition. For example, an antifreeze protein that consists essentially of a particular amino acid sequence can consist of that amino acid sequence as well as additional N- and / or C-terminal sequences that do not substantially affect the chitin decomposition activity of the enzyme.

[0026] The present invention aims to overcome the problems of current plant protection agents by providing a protein-based approach to abiotic stress and / or pest control. Specifically, the approach of the present invention relies on antifreeze proteins to protect plants. These proteins induce the activation of plant defense against stress, and some also specifically degrade chitin, which is not produced in humans or other higher animals, and are therefore expected to pose no risk to human food intake or to other non-target organisms. In this application, the term "chitinolytic enzyme" is used synonymously with the term "chitinase."

[0027] The present disclosure is described in more detail as follows.

[0028] Antifreeze Proteins The present invention relates to antifreeze proteins, compositions comprising one or more antifreeze proteins, and plant protection agents comprising or (essentially) consisting of one or more antifreeze proteins.The present invention particularly relates to the AFP of SEQ ID NO: 1 or 2, its variants or polypeptides comprising it.SEQ ID NO: 1 defines the AFP from carrot, also called Vaffr-2d.Surprisingly, the inventors have found that Vaffr-2d also has chitinolytic activity.SEQ ID NO: 2 defines the AFP named AFP-6.

[0029] An antifreeze protein as disclosed herein can comprise a first amino acid sequence that is at least 70% identical (e.g., 100% identical) to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. For example, the first amino acid sequence is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. Preferably, the first amino acid sequence is at least 98%, or at least 99% identical, most preferably 100% identical to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. That is, the protein can comprise a first amino acid sequence selected from the group of SEQ ID NOs: 1-2.

[0030] In accordance with this, a preferred antifreeze protein can also consist essentially of a first amino acid sequence that is at least 70% identical (e.g., 100% identical) to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. For example, the first amino acid sequence is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. Preferably, the first amino acid sequence is at least 98%, or at least 99% identical, most preferably 100% identical to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. That is, the protein can consist essentially of a first amino acid sequence selected from the group of SEQ ID NOs: 1-2.

[0031] Thus, the antifreeze protein can also consist of a first amino acid sequence that is at least 70% identical (e.g., 100% identical) to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. For example, the first amino acid sequence is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. Preferably, the first amino acid sequence is at least 98%, or at least 99% identical, most preferably 100% identical to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. That is, the protein can consist of a first amino acid sequence selected from the group of SEQ ID NOs: 1-2.

[0032] For example, the first amino acid sequence can be at least 70% identical to SEQ ID NO:1.

[0033] For example, the first amino acid sequence can be at least 70% identical to SEQ ID NO:2.

[0034] The antifreeze protein as disclosed herein can comprise a first amino acid sequence that exhibits up to 15 amino acid differences (e.g., no amino acid differences) with respect to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. For example, the first amino acid sequence exhibits up to 10, up to 5, or up to 3, 2, or 1 amino acid differences with respect to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. Preferably, the first amino acid sequence exhibits up to 3, 2, or 1 amino acid differences with respect to an amino acid sequence selected from the group of SEQ ID NOs: 1-2, and most preferably exhibits no amino acid differences. That is, the protein can comprise a first amino acid sequence selected from the group of SEQ ID NOs: 1-2.

[0035] In accordance with this, the antifreeze protein as disclosed herein can essentially consist of a first amino acid sequence that exhibits up to 15 amino acid differences (e.g., no amino acid differences) with respect to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. For example, the first amino acid sequence exhibits up to 10, up to 5, or up to 3, 2, or 1 amino acid differences with respect to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. Preferably, the first amino acid sequence exhibits up to 3, 2, or 1 amino acid differences with respect to an amino acid sequence selected from the group of SEQ ID NOs: 1-2, and most preferably exhibits no amino acid differences. That is, the protein can essentially consist of a first amino acid sequence selected from the group of SEQ ID NOs: 1-2.

[0036] Thus, an antifreeze protein as disclosed herein can consist of a first amino acid sequence that exhibits up to 15 amino acid differences (e.g., no amino acid differences) with respect to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. For example, the first amino acid sequence exhibits up to 10, up to 5, or up to 3, 2, or 1 amino acid differences with respect to an amino acid sequence selected from the group of SEQ ID NOs: 1-2. Preferably, the first amino acid sequence exhibits up to 3, 2, or 1 amino acid differences with respect to an amino acid sequence selected from the group of SEQ ID NOs: 1-2, and most preferably exhibits no amino acid differences. That is, the protein can consist of a first amino acid sequence selected from the group of SEQ ID NOs: 1-2.

[0037] For example, the first amino acid sequence can exhibit up to 15 amino acid differences relative to SEQ ID NO:1.

[0038] For example, the first amino acid sequence can exhibit up to 15 amino acid differences relative to SEQ ID NO:2.

[0039] When the first amino acid sequence is less than 100% identical to the reference amino acid sequence as defined above and / or has amino acid differences, the antifreeze protein preferably has the same or better protective effect (e.g. against freezing stress and / or drought stress) as the corresponding antifreeze protein consisting essentially of the reference sequence. For example, when the first amino acid sequence is at least 70% (and less than 100%) identical to the amino acid sequence of SEQ ID NO: 1, the antifreeze protein preferably has the same or better protective effect as the antifreeze protein consisting essentially of SEQ ID NO: 1. Similarly, when the first amino acid sequence has up to 15 (and at least one) amino acid differences from the amino acid sequence of SEQ ID NO: 1, the antifreeze protein preferably has the same or better protective effect as the antifreeze protein consisting essentially of SEQ ID NO: 1. The same applies mutatis mutandis to SEQ ID NO: 2.

[0040] When the first amino acid sequence is less than 100% identical to the reference amino acid sequence as defined above and / or has amino acid differences, the antifreeze protein with chitinolytic activity preferably has the same or better decomposition rate as the corresponding antifreeze protein with chitinolytic activity that consists (essentially) of the reference sequence. For example, when the first amino acid sequence is at least 70% (and less than 100%) identical to the amino acid sequence of SEQ ID NO: 1, the antifreeze protein with chitinolytic activity preferably has the same or better decomposition rate as the antifreeze protein with chitinolytic activity that consists (essentially) of SEQ ID NO: 1. Similarly, when the first amino acid sequence has up to 15 (and at least one) amino acid differences from the amino acid sequence of SEQ ID NO: 1, the antifreeze protein with chitinolytic activity preferably has the same or better decomposition rate as the antifreeze protein with chitinolytic activity that consists (essentially) of SEQ ID NO: 1.

[0041] If the first amino acid sequence is less than 100% identical to the amino acid sequence of SEQ ID NO:1 and / or has amino acid differences, the skilled artisan knows how to modify the original sequence to maintain or improve its protective effect (e.g. against freezing and / or drought stress) and / or chitin decomposition rate compared to a reference, i.e., an unmodified sequence, consisting (essentially) of an amino acid sequence. The protective effect can be determined, for example, by spraying a part of the plant with an aqueous solution of the AFP in question (or the same solution without AFP as a control) and incubating the plant at freezing temperatures (for freezing stress) or in conditions including limited water supply (for drought stress). The chitin decomposition rate can be determined, for example, by incubating chitin powder as a substrate with an aqueous solution of the AFP in question. Typically, the same method is used to determine the action of the modified enzyme and the action of the reference sequence.

[0042] The percentage of "sequence identity" or "% identical" between a first and a second amino acid sequence can be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence, and multiplying by 100%, with each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence being considered as a difference at a single amino acid residue (i.e., at a single position). The same applies mutatis mutandis to nucleotide sequences.

[0043] As used herein, "amino acid difference" can be an amino acid insertion, deletion or substitution, and is preferably a substitution. The amino acid substitution is preferably a conservative substitution as known in the art. Such a conservative substitution can be a substitution in which one amino acid in the following groups (a) to (e) is replaced by another amino acid residue in the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0044] More specifically, conservative substitutions can be as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0045] The antifreeze protein with chitinolytic activity may be an endo- or exo-chitinase. Preferably, the antifreeze protein with chitinolytic activity is capable of cleaving chitin present as a structural component of fungi and / or insects. The structural component of fungi may be the cell wall. The structural component of insects may be the exoskeleton. Most preferably, the antifreeze protein with chitinolytic activity is capable of cleaving chitin present in the cell wall of fungi.

[0046] The antifreeze protein can further comprise a second amino acid sequence fused to the N-terminus of the first amino acid sequence. The second amino acid sequence is typically located at the N-terminus of the protein.

[0047] The second amino acid sequence is preferably less than 50 amino acids in length, more preferably less than 30, even more preferably less than 25 amino acids, for example 22 amino acids.

[0048] The second amino acid sequence is typically a sequence that causes secretion from a cell, such as a bacterial cell. Thus, the second amino acid may be a signal peptide. A specific example of the second amino acid sequence is the PelB signal peptide (SEQ ID NO: 3).

[0049] The antifreeze protein can further comprise a third amino acid sequence fused C-terminally to the first amino acid sequence. The third amino acid sequence is typically located at the C-terminus of the protein.

[0050] The third amino acid sequence is preferably less than 50 amino acids in length, more preferably less than 30, even more preferably less than 20 amino acids in length. Most preferably, the third amino acid sequence is less than 10 amino acids in length, for example, 6 amino acids.

[0051] The third amino acid sequence is typically a sequence that facilitates purification of the antifreeze protein after production by a cell, such as a bacterial cell. Thus, the third amino acid can be a purification tag. Specific examples of purification tags include 6xHis tag (SEQ ID NO: 4) or Tag54 / 6xHis composite tag. Preferably, the third amino acid sequence is a 6xHis tag.

[0052] Thus, the present invention also provides an antifreeze protein comprising or (essentially) consisting of a first amino acid sequence, a second amino acid sequence and a third amino acid sequence, where the second amino acid sequence is a PelB signal peptide and the third amino acid sequence is a 6xHis tag.

[0053] The antifreeze protein is preferably a purified antifreeze protein. "Purified" in this context means that impurities are present in less than 5%, for example, impurities are present in less than 2% or even less than 1%. Impurities in this context means any substance other than protein and optionally solvent.

[0054] Particularly preferred is an antifreeze protein comprising an amino acid sequence having SEQ ID NO:1 or at least 70% sequence identity or up to 15 amino acid differences thereto.

[0055] Also particularly preferred is an antifreeze protein comprising an amino acid sequence having SEQ ID NO:2 or at least 70% sequence identity or up to 15 amino acid differences thereto.

[0056] Nucleic acids, vectors and host cells The present invention also relates to nucleic acids encoding antifreeze proteins. More specifically, the present invention provides nucleic acids encoding antifreeze proteins as described herein.

[0057] A nucleic acid encoding an antifreeze protein can also encode two or more proteins as described herein. That is, the present invention provides a nucleic acid encoding an AFP comprising a first amino acid sequence that is at least 70% identical (e.g., 100% identical) to SEQ ID NO:1 and an AFP comprising a first amino acid sequence that is at least 70% identical (e.g., 100% identical) to SEQ ID NO:2.

[0058] Nucleic acid can be, for example, DNA, RNA, or a hybrid thereof, and can also include (e.g., chemically) modified nucleotides, such as PNA. Nucleic acid can be single-stranded or double-stranded DNA. For example, the nucleotide sequence of the present disclosure can be genomic DNA, cDNA.

[0059] The present invention further provides a vector that comprises the nucleic acid that codes for antifreeze protein.As used herein, vector is a suitable vehicle for carrying genetic material into cells.Vector includes naked nucleic acid such as plasmid or mRNA, or nucleic acid embedded in larger structure such as liposome or virus vector.

[0060] A vector generally comprises at least one nucleic acid, which is optionally linked to one or more regulatory elements, such as, for example, one or more suitable promoters, enhancers, terminators, etc. The vector may be an expression vector, i.e., a vector suitable for expressing an encoded polypeptide or construct under suitable conditions, for example, when the vector is introduced into a (e.g., bacterial or plant) cell. For DNA-based vectors, this usually includes the presence of elements for transcription (e.g., promoters and polyA signals) and translation (e.g., Kozak sequences).

[0061] In a vector, the at least one nucleic acid and the regulatory element can be "operably linked" to each other, which generally means that they are in a functional relationship with each other. For example, a promoter is considered to be "operably linked" to a coding sequence if the promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of the coding sequence (in which case the coding sequence should be understood to be "under the control" of the promoter).

[0062] Also preferably, the nucleic acid encoding the antifreeze protein can be part of an expression system, where the nucleic acid represents an open reading frame, which can be codon-optimized for a particular organism.

[0063] The present invention further provides a (non-human) host or host cell comprising the nucleic acid or vector. Suitable host cells may be plant cells or microbial cells.

[0064] For example, plant cells from agricultural or ornamental plants can be used. The microbial cells can be, for example, yeast or bacterial cells, such as E. Coli. An example of a suitable yeast is Pichia pastoris.

[0065] Also provided is a plant comprising the antifreeze protein as described herein, the nucleic acid encoding it, or the vector comprising the nucleic acid.Preferably, the nucleic acid or vector can be included in the genome of the plant.Examples of plants include arable crops such as wheat, fruit-bearing plants, or vegetables.Examples of plants include cereals, corn, rapeseed, rice, soybean, or potato.

[0066] Typically, the plant (cell) will not naturally contain a gene encoding SEQ ID NO:1 or SEQ ID NO:2. For example, the plant (cell) is not Daucus carota. For example, the plant (cell) is not a green alga such as Chlorella vulgaris.

[0067] Generation method The present invention also provides a method for producing an antifreeze protein as described herein. Typically, the method comprises at least the step of culturing a host cell as described herein, in particular a bacterial host cell, such as E. coli. The culturing can be carried out in a medium suitable for the growth of the host cell.

[0068] The method may further comprise the step of harvesting the host cells and / or the culture supernatant during and / or after culturing. Preferably, the supernatant is harvested after culturing (for a suitable time).

[0069] The method can further include purifying the antifreeze protein. For example, the antifreeze protein can be purified from the culture supernatant by an initial ammonium sulfate precipitation step and sequential immobilized metal affinity chromatography purification of the solubilized protein precipitate.

[0070] Methods for producing antifreeze proteins can include, for example, constructing an expression system that includes culturing a host cell that expresses one or more antifreeze proteins.

[0071] Preferably, the antifreeze proteins produced by the method include N- and / or C-terminal modifications to facilitate secretion of the enzyme into the culture medium and / or purification from the culture supernatant. For example, the antifreeze proteins produced by the method can include a second and / or third amino acid sequence as described herein.

[0072] Thus, the method can include purifying the antifreeze protein from the culture supernatant by an initial ammonium sulfate precipitation step, followed by a sequential immobilized metal affinity chromatography purification of the solubilized protein precipitate using an amino acid tag, such as a 6xHis tag, included in the enzyme.

[0073] composition The present invention also provides a composition comprising at least one of the antifreeze proteins as described herein. Preferably, the composition induces plant immunity.

[0074] Preferably, the composition comprises at least two different antifreeze proteins as described herein.

[0075] The composition can include, for example, an antifreeze protein as described herein, including SEQ ID NO:1, and an antifreeze protein as described herein, including SEQ ID NO:2.

[0076] The composition can include, for example, an antifreeze protein as described herein consisting essentially of SEQ ID NO:1 and an antifreeze protein as described herein consisting essentially of SEQ ID NO:2.

[0077] The composition can include, for example, an antifreeze protein as described herein consisting of SEQ ID NO:1 and an antifreeze protein as described herein consisting of SEQ ID NO:2.

[0078] The composition may be a liquid or dry composition, preferably a liquid composition. The liquid composition may suitably be an aqueous composition. The composition may comprise polyvinyl alcohol (PVOH), a standard thickening agent used in commercial seed treatment. The concentration of polyvinyl alcohol in the composition may be, for example, 5% (v / v) to 15% (v / v), preferably 6% (v / v) to 10% (v / v), more preferably about 8% (v / v).

[0079] Typically, the composition does not include extracts derived from plants that naturally contain a gene encoding SEQ ID NO: 1 or SEQ ID NO: 2. For example, the composition may be a composition that does not include extracts derived from wild ginseng or green algae such as Chlorella vulgaris.

[0080] The concentration of the antifreeze protein (eg, chitinase) in the composition can be, for example, 0.01 mg / L to 250 g / L, for example, 0.025 mg / L to 100 g / L. Alternatively, the concentration of the antifreeze protein (e.g., chitinase) in the composition can be, for example, up to 1 g / L (w / v), for example up to 100 mg / L (w / v), or between 0.01 mg / L (w / v) and 100 mg / L (w / v), for example between 0.1 mg / L (w / v) and 70 mg / L (w / v), or between 1 mg / L (w / v) and 50 mg / L (w / v), or between 5 mg / L (w / v) and 30 mg / L (w / v), or between 7 mg / L (w / v) and 25 mg / L (w / v), or about 9 mg / L (w / v), or about 13 mg / L (w / v), or about 22 mg / L (w / v). Further specific examples of concentrations, depending on the application as further described herein, are as follows: For insecticidal use: 0.01% to 5% (w / v), for example 0.05% to 2.5% (w / v), preferably 0.1 to 1% (w / v) Fungicidal use: 0.25 μg / 100 μL to 25.0 μg / 100 μL, for example 0.7 μg / 100 μL to 15.0 μg / 100 μL, preferably 1.25 μg / 100 μL to 10.0 μg / 100 μL Indirect fungicidal use: 0.25 μg / 100 μL to 25.0 μg / 100 μL, for example 0.7 μg / 100 μL to 15.0 μg / 100 μL, preferably 0.65 μg / 100 μL to 5 μg / 100 μL Abiotic stress: 0.01 mg / L to 1 mg / L, for example, 0.025 mg / L to 0.5 mg / L, preferably 0.05 mg / L to 0.25 mg / L.

[0081] Preferably, the composition does not include inhibitors of protective and / or chitinolytic activity. Such inhibitors include metal ions (e.g., divalent ions, e.g., Zn 2+ , Cu 2+ , Ni 2+ ), detergents (e.g., sodium dodecyl sulfate (SDS), Triton X100 or polysorbate 20), or certain other chemicals (e.g., EDTA, imidazole). That is, for example, the composition does not contain metal ions and / or SDS.

[0082] plant protection The present inventors have surprisingly found that antifreeze proteins can be used to protect plants from various abiotic stresses, including freezing and drought stresses. Furthermore, surprisingly, AFPs, especially those with chitinolytic activity, can also be used to protect plants from harmful organisms, such as fungi.

[0083] The present inventors have also found that antifreeze proteins, particularly AFP-6 and Vaffr-2d, activate plant immune system, i.e. induce plant immunity. The terms "induce plant immunity" and "activate plant immune system" are used interchangeably herein. Induction of plant immunity can be tested, for example, by testing for activation of jasmonate pathway and / or salicylic acid pathway in plants. These pathways play an important role as regulators of plant defense against biotic and abiotic stress. Those skilled in the art are well aware of how to test for activation of jasmonate pathway and / or salicylic acid pathway. For example, activation of the jasmonate pathway can be tested by assessing changes in the mRNA levels of the LOX and / or OPR3 genes, which are upregulated to activate the jasmonate pathway (Chini, 2018: “An OPR3-independent pathway uses 4, 5-didehydrojasmonate for jasmonate synthesis”; Nature chemical biology, 14(2), 171-178; doi: https: / / doi.org / 10.1038 / nchembio.2540) (Leon, 1999: “Molecular biology of jasmonic acid biosynthesis in plants”; Plant physiology and Biochemistry, 37(5), 373-380; doi: https: / / doi.org / 10.1016 / S0981-9428(99)80043-6). Activation of the salicylic acid pathway can be tested by assessing changes in the mRNA levels of the PR1-3 and / or PR1-17 genes.The production of PR1-3 and PR1-17 proteins is associated with the plant hormone salicylic acid (Van Loon, 1999: "The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins"; Physiological and molecular plant pathology, 55(2), 85-97; doi: https: / / doi.org / 10.1006 / pmpp.1999.0213). Exemplary methods for testing for activation of the jasmonate and / or salicylic acid pathways are further described in Example 7 below. The plant immune system is conserved in all plants, and therefore it can be assumed that the antifreeze protein of the present invention activates the immune system in all plants. Advantageously, plant immunity is induced before the plant is damaged by abiotic stress and / or pests. Thus, preferably, the antifreeze protein of the present invention (or a composition comprising it) is applied to a plant or a part thereof before abiotic stress and / or pests occur, e.g., before the plant is damaged by abiotic stress and / or pests.

[0084] Furthermore, the inventors have found that the application of the antifreeze protein according to the invention can be combined with the application of a plant protection agent of the prior art, thereby further enhancing plant protection. Thus, a further plant protection agent that is not an antifreeze protein can be applied to the plant or a part thereof in addition to the application of the antifreeze protein according to the invention. Such further plant protection agents can be, for example, chemical insecticides, such as substances from the groups of organochlorines, organophosphates, carbamates, pyrethroids, triazines and neonicotinoids, for use as insecticides, herbicides, fungicides and rodenticides. For example, such further plant protection agents can be Difend extra (25 g / L difeconazole and 25 g / L fludioxinil) and / or Protendo (prothioconazole 300 g / L EC) and / or Velogy era (EC 75 g / L benzovindiflupyr + 150 g / L prothioconazole). Such further plant protection agents can also be, for example, the further agents ascorbic acid, betaine and / or salicylic acid mentioned below.

[0085] Apart from their action against pests and abiotic stresses by inducing natural defense mechanisms in plants against fungal and abiotic stresses, the chitinolytic activity of antifreeze proteins may also have a direct effect against pests and plant diseases.

[0086] That is, the present invention provides a composition comprising at least one antifreeze protein as described herein that is a plant protection agent. The present invention further provides a composition comprising at least two antifreeze proteins as described herein that is a plant protection agent.

[0087] The present invention also provides for the use of a composition comprising at least one antifreeze protein as described herein as a plant protection agent.The present invention also provides for the use of a composition comprising at least two antifreeze proteins as described herein as a plant protection agent.

[0088] The plant protection agent is preferably for protecting the plant against abiotic stresses and / or for protecting the plant against pests, for example against chitin-containing organisms.

[0089] That is, the plant protection agent can be for protecting a plant against abiotic stress. Abiotic stress includes, for example, freezing stress, drought stress, salt stress, waterlogging stress or heat stress. Preferably, the abiotic stress is freezing stress or drought stress.

[0090] The plant protection agent may (also) be for protecting plants against harmful organisms, for example against organisms that contain chitin. For example, chitin is the main component of the cell walls of fungi, the exoskeletons of arthropods such as insects, and the radula of mollusks. Thus, the plant protection agent may be against the infestation of fungi, insects or mollusks, preferably fungi or insects, most preferably fungi.

[0091] Examples of fungi include, for example, ascomycetes from the Nectriaceae or Mycosphaerellaceae families. Examples of fungi from the Nectriaceae family include fungi from the genus Fusarium, such as Fusarium oxysporum, Fusarium graminearum, and Fusarium culmorum. Examples of fungi from the Mycosphaerellaceae family include fungi from the genus Septoria, such as Septoria tritici. Other examples include Alternaria solani, Phytophtora infestans, Pythium, Magnaporthe oryzae, Venturia inaequalis, Pyrenophora teres, Rhynchosporium secalis, Puccinia triticina, and Ramularia collo-cygni. The fungus may be a filamentous fungus. The fungus is typically a pathogenic fungus.

[0092] Examples of insects include insects from the Aphididae, Tenebrionidae, Drosophilidae or Aphrophoridae families. Examples of insects from the Aphididae family include insects from the Sitobion genus, such as Sitobion avanae. Examples of insects from the Tenebrionidae family include insects from the Tribolium genus, such as Tribolium castaneum. Examples of insects from the Drosophilidae family include insects from the Drosophila genus, such as Drosophila melanogaster. Examples of insects from the family Spirulina include insects from the genus Philaenus, such as Philaenus spumarius.

[0093] The plants to be protected are not particularly limited, and examples thereof include arable crops such as wheat, fruit-bearing plants, and vegetables.

[0094] Examples of plants include cereals, maize, rapeseed, rice, soybean or potato.

[0095] The present invention further provides a method for protecting a plant from abiotic stress and / or pests, comprising application of an antifreeze protein, such as an antifreeze protein as described herein, or a composition comprising at least one antifreeze protein, to the plant or a part thereof. Typically, the composition is applied to the surface of the plant or a part thereof.

[0096] Examples of abiotic stress include freezing stress, drought stress, salt stress, and heat stress. Preferably, the abiotic stress is freezing stress or drought stress.

[0097] Pests include, for example, fungi, insects or mollusks. Examples are given above. Preferably, the pest is a fungus or insect, most preferably a fungus. Pests can be biotrophic or necrotrophic.

[0098] Application of the antifreeze protein or composition can include, for example, immersing the plant or plant part in the composition as described herein. Another exemplary application of the antifreeze protein can include coating the plant or plant part (e.g., seed) with the antifreeze protein, for example, using the composition as described herein, for example, using the coating method described in the Examples. Another exemplary application of the antifreeze protein can include spraying the composition as described herein onto the plant or plant part. For example, the antifreeze protein can be sprayed at a dose rate of about 70 mg / ha using about 200 L of water per hectare (i.e., using a composition that includes the antifreeze protein at a concentration of about 0.35 mg / L), or using 1000 L or 1500 L of water / ha.

[0099] The plant part can be, for example, a leaf, a fruit or a seed (such as a grain). When the plant part is a seed, the antifreeze protein of the present invention (or a composition containing it) can be preferably applied to the seed before sowing. The seed can be a coated seed or a non-coated seed. Coating seed technology is generally known to those skilled in the art and can be easily modified.

[0100] The plants to be protected are not particularly limited and may be, for example, arable crops, fruit-bearing plants or vegetables. Examples are described above.

[0101] Plant protection can also be achieved by expressing at least one antifreeze protein as described herein in plant or plant cell.Thus, the present invention also provides the use of nucleic acid or vector as described herein for expressing antifreeze protein in plant or plant cell.Expression can be constitutive or inducible.For example, expression can be inducible in response to external stimuli, such as abiotic stress and / or pest infestation (e.g., via endogenous sensory mechanisms in plants that can detect abiotic stress and / or tissue damage).

[0102] Further drugs The antifreeze proteins can be suitably combined with further agents that can act as plant protection agents, such as agents against abiotic stress, fungicides and / or insecticides. Such further agents can be, for example, ascorbic acid, betaine and / or salicylic acid.

[0103] Thus, the present invention also provides compositions comprising at least one antifreeze protein as described herein, further comprising ascorbic acid, betaine and / or salicylic acid, and uses thereof.

[0104] The present invention also provides compositions comprising at least one antifreeze protein as described herein, and uses thereof, which further comprise a fungicide and / or an insecticide.

[0105] [Table 1] EXAMPLES

[0106] The following experimental section of this application relates to non-limiting exemplary embodiments of the present invention.

[0107] Example 1 The protective activity of antifreeze proteins against fungal infestation was tested. For this purpose, antifreeze proteins were produced in E. coli BL21 cells. The enzymes were purified from the culture supernatant.

[0108] Antifreeze proteins were tested for their protective activity in a germination test. Briefly, the following steps were performed: 1. Sterilization of wheat seeds using 10% bleach for 10 min in a safety cabinet 2. Germination on filter paper 3. Foliar spray 3 dps (days after sowing) and 1 dbi (days before infection) 4. 4dps: Add the pathogen Fusarium culmorum 5. Germination and plant health / phytotoxicity monitoring.

[0109] The results are shown in Figure 1. The results show that addition of the antifreeze protein Vaffr-2d with chitinolytic activity led to a strong inhibition of fungal growth and improved plant health (biostimulant effect compared to the control).

[0110] In other words, these results show that antifreeze proteins with chitinolytic activity, particularly the antifreeze proteins described herein, can effectively inhibit the growth of harmful organisms such as fungi on plants, thereby improving plant health. Thus, the antifreeze proteins can be used as plant protection agents as described herein.

[0111] Example 2 The protective activity of antifreeze proteins against freezing stress was tested by foliar spraying of antifreeze protein Vaffr-2d (200 L / ha aqueous formulation containing antifreeze proteins at the indicated concentrations) on apple plants and subjecting the plants to freezing stress. The results are shown in Table 2 below and in Figure 2.

[0112] [Table 2]

[0113] The results show that the addition of Vaffr-2d resulted in reduced frost damage and improved plant health with increasing dosage.

[0114] In other words, these results show that antifreeze proteins, particularly the antifreeze proteins described herein, can effectively reduce frost damage, which can improve plant health. That is, antifreeze proteins can be used as plant protection agents as described herein.

[0115] Example 3 The protective activity of antifreeze proteins against freezing stress was tested by foliar spraying of antifreeze proteins Vaffr-2d or AFP-6 on pear plants and subjecting the plants to freezing stress. The results are shown in FIG.

[0116] The results show that addition of Vaffr-2d or AFP-6 resulted in increased pear yield compared to the control.

[0117] In other words, these results show that antifreeze proteins, particularly the antifreeze proteins described herein, can effectively reduce frost damage, which can improve plant health. That is, antifreeze proteins can be used as plant protection agents as described herein.

[0118] Example 4 Different plants were subjected to different kinds of abiotic stress with or without foliar spray of Vaffr-2d or AFP-6.

[0119] Set up for salt stress experiment: Sow the plants in rows in the trays Germination at 25℃ / 15℃ (day / night) 16 hours light, 8 hours dark Application after 10 days After 2 days, replant in an 8x8cm pot. 3 possible regimes * Direct salt stress at the moment of replanting * Salt stress 4 days after repotting * Salt stress 7 days after replanting Salt stress = 100 mL of 120 g / L NaCl solution.

[0120] The results are shown in Table 3 below.

[0121] [Table 3]

[0122] These results show that antifreeze proteins, particularly the antifreeze proteins described herein, can effectively protect plants from various abiotic stresses, such as salt stress, drought stress or waterlogging stress. This can improve plant health. That is, antifreeze proteins can be used as plant protection agents as described herein.

[0123] Example 5 To investigate the protective activity of the enzyme AFP-6 against fungal infection, two greenhouse experiments were performed.

[0124] For both tests, seeds were coated in a Satec ML2000, 8 L / ton coater according to the following steps (which can be extrapolated to, for example, commercially available equipment): - Required amount of seeds in the batch coater (min 50g and max 2kg in Satec ML2000) - Medication start: takes about 10 to 15 seconds - Then, leave the device running for a further 15 seconds to dry. - Remove from device - Rotor speed: 500~1000RPM - Rotation speed of rotating disc: 2100~3000RPM - Coating temperature during the whole process: ±20℃ - The coating process involves loading, dosing, drying, and unloading, which takes approximately 1 minute in total.

[0125] Coating was performed with 0.07 g AFP-6 (produced by Fraunhofer-Gesellschaft) in 8 L water with 8% (v / v) PVOH per ton of seeds (i.e. in 7.36 L water + 640 mL PVOH). The final concentration of AFP-6 after coating was therefore 0.07 g / ton of seeds. Polyvinyl alcohol (PVOH) is a standard thickener used in commercial seed treatments. The untreated control was water and 8% PVOH only, no active ingredient was added. Four seeds were sown per pot in six replicates. In a second test, seeds were alternatively coated with Difend extra (25 g / L difeconazole and 25 g / L fludioxinil) at a concentration of 2 L / ton for comparison. Difend extra is a commercially available seed treatment.

[0126] The first test was carried out with the wheat cultivar Benchmark. Two weeks after sowing, heavily infected wheat plants with red rust (infected with Puccinia triticina) were placed in the test to mimic natural infection. After 8 days, the percentage of red rust spots in treated and untreated controls was determined for the three upper leaves of the plants. The results of the enzyme-treated controls were compared to the untreated control and the percentage effectiveness of the enzyme compared to the untreated control was calculated based on the percentage of infection. The youngest leaf (leaf 1) was not infected in the treated controls and the untreated control. Therefore, leaves 2 and 3 were evaluated. Leaf 2 was 69% less infected and leaf 3 was 44% less infected compared to the untreated control (see Figure 4). This is an average effectiveness of 57% less infection for both leaves for the cultivar Benchmark coated with 0.07 g / ton of the enzyme AFP-6.

[0127] For the second test, the wheat cultivar Keitum was used. Two weeks after sowing, the plants were inoculated with a suspension of red rust spores (3 × 10 5 The plants were inoculated with 0.001 spores / mL of 1000 spores per mL until runoff. The percentage of red rust spots was assessed 12 days after infection as described above for the four upper leaves. The results of AFP-6 or Difend extra treated subjects were compared to untreated controls and the percentage efficacy of the treated subjects compared to the untreated control was calculated based on the percentage of infection. The youngest leaf (leaf 1) was not infected in the treated subjects and the untreated control. Therefore, leaves 4-3 and 2 were scored for red rust. Comparing AFP-6 treated plants to the untreated control, leaf 2 was 100% less infected and leaf 3 was 32% less infected. The oldest leaf (4) was 27% more protected. This was an average efficacy of 31% for all leaves on cultivar Keitum coated with 0.07 g / ton of the enzyme AFP-6 (see Figure 5A). No or only slight protection was observed for seeds coated with Difend extra, even though much higher concentrations were used (see FIG. 5B).

[0128] These results showed that seed treatment with AFP-6 protected plants against fungal infection by Puccinia triticina, and that the protection provided by AFP-6 was much better than that provided by the commercial seed treatment Difend extra. Because protection was achieved by treating seeds with AFP-6 (i.e., by indirect fungicidal application), the AFP-6 concentrations used were very low, and relatively young leaves were better protected than older leaves, these results suggested that the enzyme AFP-6 exerted a protective effect by inducing plant immunity ("indirect fungicidal effect") against both wheat cultivars tested.

[0129] Example 6 Two further tests were carried out to investigate whether the enzyme AFP-6 could further improve plant protection against foliar diseases when plants were treated with AFP-6 in addition to standard chemical treatments. These tests were carried out in practical field conditions, where wheat was treated by farmers with chemical seed treatments (Difend extra at a concentration of 2L / ton, 25g / L difeconazole and 25g / L fludioxinil) and at least two foliar treatments with chemicals were carried out during the season: T1=BBCH 32 and T2=BBCH 39. For T1 Protendo, prothioconazole 300g / L EC (400mL / ha) was used, and for T2 Velogy era, EC 75g / L benzovindiflupyr + 150g / L prothioconazole (1000mL / ha) was used.

[0130] Since previous experiments suggested that the enzyme AFP-6 exerted a protective effect by inducing plant immunity, in both trials the enzyme AFP-6 was added as early in the season as possible and then the plants were infected with the fungal pathogen. The trials were carried out at 400 seeds / m 2 Each plot was 24 m 2 and all plots had 4 replicates. In each plot, 4 plants were evaluated according to EPPO guideline PP(1) / 026(4) for foliar disease and for cereals for ear disease. The infection percentage of the leaf disease was scored on the 3 upper leaves of the plant. Results: abbott. The average infection percentage of the 16 plants was calculated (4 plants / plot). The percentage of efficacy of pest severity was calculated as a function of the subjects chemically coated with Difend extra (for the 1st test as described below) or without T0 treatment (for the 2nd test as described below).

[0131] In the first test, the enzyme AFP-6 was used as a seed treatment at a concentration of 0.07 g / ton. The variety in this test was Keitum. The control was coated with the chemical seed treatment Difend extra (25 g / L difeconazole and 25 g / L fludioxinil) at a concentration of 2 L / ton. Seeds were coated in a Satec ML2000, 8 L / ton coater as described for Example 5 above. Coating with the enzyme was performed with 0.07 g AFP-6 (produced by Fraunhofer-Gesellschaft) in 8 L of water with 8% (v / v) PVOH per ton of seed (i.e. in 7.36 L water + 640 mL PVOH). Thus, the final concentration of the enzyme after coating was 0.07 g / ton of seed. Polyvinyl alcohol is a standard thickener used in commercial seed treatments. Foliage disease was assessed 169 days after sowing and compared with controls coated with the chemical Difend extra.

[0132] A second trial investigated whether an earlier treatment with AFP-6 prior to BBCH 32 (i.e. a "T0" treatment at BBCH 30) could further improve plant protection against foliar disease. Cultivar Ragnar was used in this trial. Seeds were given a chemical seed treatment with defend extra. Plants were sprayed using a concentration of 0.07 g / L AFP-6 in a water formulation using 1 L / ha. Foliar treatments were given at BBCH 30. Plants were assessed for foliar disease 13 days after T0 treatment and compared to controls that did not receive a T0 treatment.

[0133] Both tests were evaluated on the same day. Only leaf 3 showed infection by the fungus Septoria tritici, which causes a foliar disease. In both tests it was observed that the enzyme AFP-6 improved plant protection. As a seed treatment at a concentration of 0.07 g / ton seed (first test) in the cultivar Keitum, the efficacy of the enzyme AFP-6 was 24% compared to the chemical seed treatment Difend extra. In the cultivar Ragnar (second test), the T0 treatment with BBCH 30 showed 25% efficacy compared to no T0 treatment. These results obtained with low concentrations of AFP-6 confirm that AFP-6 exerts a protective effect by inducing plant immunity.

[0134] Example 7 Further tests were performed to confirm that AFP-6 and Vaffr-2d exert their protective effect by inducing plant immunity. In this regard, it is important to note that different plant immunity pathways can be elicited depending on the type of stress to which the plant is exposed: The plant hormones jasmonic acid and salicylic acid play important roles as regulators of plant defense against biotic and abiotic stresses. Their effects on plant immunity against biotrophic pathogens depend on salicylic acid. With regard to necrotrophic pathogens, jasmonic acid is more important (Glazebrook, 2005: “Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens”; Annual review of phytopathology, 43, 205; doi: doi. 10.1146 / annurev.phyto.43.040204.135923). Different types of abiotic stress will also trigger different pathways in plants (Peleg, 2011: “Hormone balance and abiotic stress tolerance in crop plants”; Current opinion in plant biology, 14(3), 290-295; doi: https: / / doi.org / 10.1016 / j.pbi.2011.02.001).

[0135] Therefore, for the following studies, four different genes were selected to demonstrate that AFP-6 and Vaffr-2d have an effect on plant immunity, namely, LOX, OPR3, PR1-3 and PR1-17 genes. LOX and OPR3 genes are upregulated to activate the jasmonate pathway (Chini, 2018: “An OPR3-independent pathway uses 4, 5-didehydrojasmonate for jasmonate synthesis”; Nature chemical biology, 14(2), 171-178; doi: https: / / doi.org / 10.1038 / nchembio.2540) (Leon, 1999: “Molecular biology of jasmonic acid biosynthesis in plants”; Plant physiology and Biochemistry, 37(5), 373-380; doi: https: / / doi.org / 10.1016 / S0981-9428(99)80043-6). As shown above, jasmonate is an important plant hormone that regulates plant immunity against necrotrophic pathogens. PR1-3 and PR1-17 are PR proteins produced by plants to defend them against biotrophic pathogens. The production of these compounds is associated with the plant hormone salicylic acid (Van Loon, 1999: "The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins"; Physiological and molecular plant pathology, 55(2), 85-97; doi: https: / / doi.org / 10.1006 / pmpp.1999.0213).

[0136] AFP-6 Laboratory tests were conducted to investigate the plant immunity induction potential of AFP-6. Following the steps outlined in Example 5 above, wheat (variety chamsin) was coated with two concentrations of AFP-6 in a Satec ML2000, 8L / ton coater. Coating was performed with 0.1g or 0.175g of AFP-6 (produced by Fraunhofer-Gesellschaft) in 8L of water with 8% (v / v) PVOH per ton of seed (i.e., in 7.36L water + 640mL PVOH). Thus, the final concentration of the enzyme after coating was 0.1g / ton and 0.175g / ton seed. PVOH is a standard thickener used in commercial seed treatments. An untreated control was coated with water and 8% PVOH. Five seeds were placed on moister filter paper in a petri dish. Seeds were germinated in a 20°C incubator with a 12-h light / 12-h dark regime. For each subject, three replicates were performed for each sampling time point. Seedling samples were taken at different times after "seeding" on filter paper: 4-7-9 and 14 days.

[0137] Total mRNA was isolated from plant tissues using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA). Total RNA concentration was quantified using the NanoDrop method (ND-1000 spectrophotometer, Thermo Scientific, USA). cDNA was generated from 1 mg of RNA of each sample by using anchored oligo(dT)18 and hexamer primers according to the instructions of the Transcriptor First-Strand cDNA Synthesis Kit (Roche). cDNA was generated using StepOnePlus qPCR Mastermix Plus for SYBR Green I (Eurogentec, San Diego, CA, USA) with primers targeting the gene of interest. TMqRT-PCR was performed on a PCR machine (Applied Biosystems, UK). Expression levels of transcripts from different samples were normalized to the normalized reference genes actin and ubiquitin, and PCR data were analyzed using the 2-ΔCt method.

[0138] The results are shown in Figure 6. These results indicate that AFP-6 upregulates both the jasmonic acid pathway (LOX and OPR3; see Figures 6A and 6B, respectively) and the salicylic acid pathway (PR1-3 and PR1-17; see Figures 6C and 6D, respectively). This means that AFP-6 primes plant immunity against both necrotrophic and biotrophic pathogens (i.e., against various types of pests) as well as various types of abiotic stresses.

[0139] Vaffr-2d Greenhouse trials were conducted to investigate the plant immunity induction potential of Vaffr-2d. Following the steps outlined in Example 5 above, seeds were coated in a Satec ML2000, 8L / ton coater with one concentration of Vaffr-2d for wheat (variety chamsin). Coating was performed with 0.175g of Vaffr-2d in 8L of water with 8% (v / v) PVOH per ton of seed (i.e., in 7.36L water + 640mL PVOH). Thus, the final concentration of enzyme after coating was 0.175g / ton seed. PVOH is a standard thickening agent used in commercial seed treatments. An untreated control was coated with water and 8% PVOH. 40 seeds / pot were sown in potting soil, with 4 pots. Plant samples were taken at different time points after sowing: 6-7-8 and 11 days. Extraction of mRNA and qRT-PCR were performed as described above for AFP-6. The same genes were selected as in the AFP-6 study.

[0140] The results are shown in Figure 7. These results show that Vaffr-2d also upregulates both the jasmonic acid pathway and the salicylic acid pathway. These results indicate that, for example, Vaffr-2d can be used as a preventative treatment to prime plant immunity against a variety of biotic and abiotic stresses. [Industrial Applicability]

[0141] The antifreeze proteins described herein, particularly those for use in plant protection, as well as the related products and uses described herein, can be applied, for example, to commercial plant protection agents, for example, for use in agriculture. Thus, the present disclosure is industrially applicable.

Claims

1. A composition comprising at least one antifreeze protein comprising a first amino acid sequence that is at least 70%, for example 100%, identical to an amino acid sequence selected from the group of SEQ ID NOs: 1-2.

2. The composition according to claim 1, wherein the antifreeze protein comprises the amino acid sequence set forth in SEQ ID NO:

1.

3. The composition according to claim 1, wherein the antifreeze protein comprises the amino acid sequence set forth in SEQ ID NO:

2.

4. The composition according to any one of claims 1-3, which is a plant protection agent.

5. The composition according to any one of claims 1-3, wherein the antifreeze protein is contained in the composition at a concentration of 0.01 mg / L (w / v) to 100 mg / L (w / v).

6. The composition according to claim 4, wherein the plant protection agent is against abiotic stress, and the antifreeze protein is contained in the composition at a concentration of 1 mg / L (w / v) to 100 mg / L (w / v).

7. Use of a composition comprising at least one antifreeze protein as a plant protection agent.

8. The use according to claim 7, wherein the composition is as described in any one of claims 1-3.

9. The use according to claim 7 as a plant protection agent against abiotic stress and / or pests.

10. The use according to claim 7, wherein the plant protection agent is against abiotic stress, and the abiotic stress is optionally freezing stress or drought stress.

11. The use according to claim 7, wherein the plant protection agent is against pests, and the pests are optionally organisms containing chitin such as fungi or insects.

12. Use of the composition according to claim 7, wherein the plant protection agent is against pests and the pests are fungi.

13. Use according to claim 11, wherein the fungus is a Fusarium or Septoria species.

14. Use according to claim 7, wherein the plant is a field crop, a fruiting plant or a vegetable.

15. Use according to claim 7 for inducing plant immunity.

16. A method for protecting a plant from abiotic stress and / or pests, comprising applying a composition comprising at least one antifreeze protein to the plant or a part thereof.

17. The method according to claim 16, wherein the composition is as described in any one of claims 1 to 3.

18. The method according to claim 16, wherein the plant or plant part is immersed in the composition.

19. The method according to claim 16, wherein the composition is applied by spraying onto the surface of a plant, or a part of a plant such as a coated seed.

20. The method according to claim 16, wherein the method is for protecting a plant from abiotic stress, and the abiotic stress is optionally freezing stress or drought stress.

21. The method according to claim 16, wherein the pest is a chitin-containing organism such as a fungus or an insect.

22. The method according to claim 16, wherein the pest is a fungus.

23. The method according to claim 21, wherein the fungus is a Fusarium or Septoria species.

24. The method according to claim 16, wherein the plant is an arable crop, a fruiting plant or a vegetable.

25. The method according to claim 16 for inducing plant immunity.

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