Compositions for delivering agents to plant cells

Abstract

Described are peptides comprising a membrane translocation domain having one or more cell penetrating peptide motifs and a cargo moiety linked to the membrane translocation domain, wherein the cargo moiety comprises a plant bioactive moiety. The at least one cell penetrating peptide motif has a length of 3 to 10 amino acid residues and has at least three arginine and / or lysine residues; or the at least one cell penetrating peptide motif has a length of from 3 to 10 amino acid residues and has at least two arginine and / or lysine residues and the at least one other cell penetrating peptide motif has a length of from 2 to 8 amino acid residues and has at least two hydrophobic residues. Also described are methods of delivering cargo moieties into plant cells, comprising contacting the plant cells with the peptides disclosed herein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 338,302, filed May 4, 2022, which is hereby incorporated by reference in its entirety. References to sequence listings

[0002] Pursuant to 37 CFR § 1.52(e)(5), the sequence listing submitted on May 4, 2023 is incorporated herein by reference as a text file named “103361-267WO1_ST26” created on May 4, 2023 and having a file size of 295,920 bytes. Background Art

[0003] Although fertilizers and pesticides play a vital role in modern agriculture, their adverse effects on the environment and animal / human health have recently prompted the rapid rise of organic agriculture. It is clear that organic agriculture, with a market of approximately $80 billion, will become an essential innovative agricultural system that balances sustainability with food / ecosystem security and human health benefits (Reganold and Wachter, 2016). One promising environmentally friendly innovation is the use of (i) biodefense activators that improve plant immunity against pathogens and insects, and (ii) biostimulants that enhance plant growth and tolerance to abiotic stresses. Many of the plant defense activators and biostimulants currently used in organic agriculture are proteins / peptides that can trigger appropriate signal transduction pathways to stimulate defense and / or growth. However, a big challenge in the use of these peptides / proteins is their poor penetration efficiency in foliar applications and seed treatments (Nadendla, SR, et al., Carbohyd Polym 199, 11-19 (2018)).

[0004] Cell penetrating peptides (CPPs) were first discovered in the early 1990s (Vives, E., et al., J Biol Chem 272, 16010-16017 (1997) and Derossi, D., et al., J Biol Chem 269, 10444-10450 (1994)). Since then, nearly 2000 CPPs have been reported, most of which are linear peptides. Despite much effort by academia and industry, drug delivery using linear CPPs has been largely unsuccessful because they are unstable upon proteolysis and exhibit low cytosolic entry efficiency and poor pharmacokinetics. Cyclization of CPPs has produced an important advance because cyclic variants are stable upon proteolysis and exhibit a 60-fold increased cytosolic entry efficiency compared to their linear counterparts (Qian, Z. et al. Biochemistry 55, 2601-2612 (2016)). Further studies have shown that the powerful activity of cyclic CPPs is due to their ability to escape endosomes through a previously unappreciated vesicle budding and collapse pathway (Sahni, A., et al., ACS Chem Biol 15, 2485-2492 (2020)). This understanding, in turn, has led to the realization that CPPs must adopt appropriate 3-D structures to achieve high cell penetrating activity. Based on this insight, Bhat et al. recently engineered MTD4, a highly potent CPP derived from the tenth human fibronectin type III (FN3) domain. Unlike cyclic CPPs that must be chemically synthesized before conjugation to proteins, MTD4 can be genetically fused to any peptide / protein cargo and recombinantly produced.

[0005] Research on CPP-mediated plant protein delivery began 15 years ago. The first study showed that CPP can be internalized into tobacco protoplasts, indicating that these peptides can enter plant cells by transfection (Mäe, M., et al. Biochimica et Biophysica Acta (BBA)-Biomembranes 1669, 101-107 (2005)). Subsequently, it was also reported that various CPPs were translocated in immature embryos of wheat in the presence of cell membrane permeabilizing agents (Chugh, A., et al., FEBS J 275, 2403-2414 (2008)). The successful uptake of protein cargo by living microspore cells is also achieved by utilizing the reversible disulfide bond between R9 CPP and mCherry protein (Bilichak, A., et al., Front Plant Sci 6 (2015)). Recently, the penetration efficiency of 55 CPPs, most of which had been previously tested in animals, was evaluated in dicots and monocots, and some were found to enter plant cells (Numata, K., et al., SCI REP-UK 8 (2018)). A study of the delivery efficiency of two CPPs into rice callus revealed that 5-day-old callus was more suitable for CPP uptake than 21-day-old callus (Guo, B., et al., PLOS ONE 14, e214033 (2019)). Although many CPPs have been tested in plants, there are no reports of using CPPs to deliver defense or growth-promoting proteins / peptides to crop plants.

[0006] The compositions and methods disclosed herein address these and other needs. Summary of the invention

[0007] Compounds, compositions, methods for preparing and using such compounds and compositions are disclosed herein. In one aspect, a peptide comprising a membrane translocation domain having one or more cell-penetrating peptide motifs and a cargo portion connected to the membrane translocation domain is disclosed, wherein the cargo portion includes a plant biologically active portion, wherein at least one cell-penetrating peptide motif has a length of 3 to 10 amino acid residues and has at least three arginine and / or lysine residues; or wherein at least one cell-penetrating peptide motif has a length of 3 to 10 amino acid residues and has at least two arginine and / or lysine residues, and at least one other cell-penetrating peptide motif has a length of 2 to 8 amino acid residues and has at least two hydrophobic residues. A method for delivering a plant biologically active portion into a plant cell is also disclosed, comprising contacting a plant cell with a peptide disclosed herein.

[0008] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and, together with the description, serve to explain the principles of the invention.

[0010] Figure 1 The structures of WT FN3 and MTD1-5 are shown. The FN3 structure was generated from PDB file 1ttg and the structures of MTD1-5 were predicted by Phyre2. The inserted MTD1-5 CPP motif is highlighted in black.

[0011] Figure 2A and 2B . Shown is the expression and purification of MTD4. Figure 2A is an FPLC chromatogram showing the elution of MTD4 from a Ni-NTA column (MTD4 is eluted as a broad peak); Figure 2B is an SDS-PAGE showing the expression level and different fractions during purification on a Ni-NTA column. L, molecular weight marker; U, crude lysate of uninduced cells; I, crude lysate of IPTG-induced cells; CL, crude cell lysate after centrifugation; FT, flow-through fraction; 1-10, Ni-NTA column elution fractions (strong band is MTD4).

[0012] Figure 3A-3B . (3A) MTD4 structure is shown. (3B) Permeability of MTD4 protein in N. benthamiana leaves. Two days after GFP permeabilization, 10 µM rhodamine-labeled MTD4 (MTD4-Rh) or FN3 (FN3-Rh) was sprayed on N. benthamiana leaves. Two hours after spraying, H 2 O 2 The sprayed leaf areas were washed to remove any residual protein and then red (Rh) and green fluorescence (GFP) were measured using confocal microscopy.

[0013] Figures 4A-4G. MTD4 was shown to enhance the permeability and disease resistance of HrpZ in tobacco and tomato. (4A) are images showing cell death caused by MTD4-HrpZ treatment. 2 ml of 10 µM MTD4-HrpZ, HrpZ, or MTD4 recombinant protein were placed on leaves of 6-week-old Nicotiana benthamiana plants. The images shown were taken 24 hours after treatment with the respective proteins. (4B) are images showing the accumulation of reactive oxygen species (ROS) after MTD4-HrpZ treatment. MTD4-HrpZ, HrpZ, or MTD4 (10 µM) were sprayed on leaves of 1-month-old Nicotiana benthamiana plants. ROS accumulation was detected by 3,3'-diaminobenzidine (DAB) staining. The images were taken 12 hours after treatment with the respective proteins. (4C-4D) are graphs showing qRT-PCR analysis of the expression levels of (4C) NtHSR203 and (4D) NtCHN50 after treatment with MTD4-HrpZ, HrpZ, or MTD4 proteins. Letters (a, b, c) indicate significant differences (P < 0.05, Dunnett's multiple range test). (4E) is a graph showing the enhanced resistance of plants treated with MTD4-HrpZ. MTD4-HrpZ, HrpZ, or MTD4 proteins (2 µM) were sprayed on N. benthamiana leaves. 12 h after spraying, the leaves were pressure inoculated with Pst DC3000 hrcC-, and the OD values ​​were 600 About 0.2. The number of bacteria in plants was evaluated at 0 and 3 days after inoculation and expressed as colony forming units (cfu). The data presented are mean ± standard error; a and b indicate statistically significant differences (Duncan multiple range test, P < 0.05). (4F) is an image showing disease symptoms of tomatoes treated with MTD4-HrpZ, HrpZ or MTD4 after inoculation with Botrytis cinerea. MTD4-HrpZ, HrpZ or MTD4 protein (5 µM) was sprayed on the disinfected tobacco surface. Blocks of Botrytis cinerea hyphae were placed on small wound sites of 24-hour protein-treated tomato fruits. The photos were taken at 3 dpi. (4G) is a graph showing the diameter of the infection area measured at 3 dpi.

[0014] Figure 5 . are images showing cell death caused by MTD4-HrpZ in Arabidopsis thaliana after spraying on leaves of 6-week-old plants. Images were taken 24 hours after treatment.

[0015] Figures 6A-6C are images showing that MTD4-HrpZ confers resistance to tomato bacterial spot pathogen. (6A) Image after application of MTDA4, 1 μM, (6B) Image after application of HrpZ, 1 μM, and (6C) Image after application of MTD4-HrpZ, 1 μM.

[0016] Figures 7A-7D are images showing that MTD4-HrpZ confers resistance to tomato bacterial spot pathogen. (7A) is an image after application of MTD4; (7B-7D) are images after application of MTD4-HrpZ at different cell lysate dilutions: (7B) 3-fold diluted cell lysate, (7C) 4-fold diluted cell lysate, and (7D) 5-fold diluted cell lysate.

[0017] Figure 8 Images showing that MTD4-HrpZ promotes tomato growth. Plants were sprayed twice with the three proteins, and the data were obtained from a two-repeat experiment.

[0018] Figures 9A-9D 9A) is a graph showing that MTD4-HrpZ promotes tomato growth. (9B) is a graph showing plant height up to the shoot tip for MTD4, HrpZ, and MTD4-HrpZ. (9C) is a graph showing fresh weight (g) for MTD4, HrpZ, and MTD4-HrpZ. (9D) is a graph showing dry weight (g) for MTD4, HrpZ, and MTD4-HrpZ. Letters (a, b, c) indicate significant differences (P < 0.05, Dunnett's multiple range test). DETAILED DESCRIPTION

[0019] The present invention may be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0020] Before disclosing and describing the compounds, compositions, articles, systems, devices and / or methods of the present invention, it should be understood that unless otherwise indicated, they are not limited to specific synthetic methods, or unless otherwise indicated, they are not limited to specific reagents, and therefore may of course be different. It should also be understood that the terms used herein are only for the purpose of describing specific aspects and are not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein can be used to practice or test the present invention, the methods and materials of the embodiments are now described.

[0021] All publications mentioned herein are incorporated herein by reference, to disclose and describe methods and / or materials relevant to quoting the publication. The publication discussed herein is provided only for its disclosure before the filing date of the present application. This article does not have any content to be interpreted as admitting that the present invention is not qualified to precede this publication of existing invention. In addition, the publication date provided herein may be different from the actual publication date, which may need to be confirmed separately. definition

[0022] In this specification, the terms "about" and / or "approximately" may be used in conjunction with numerical values ​​and / or ranges. The term "about" should be understood as values ​​close to the stated values, as well as the stated values.

[0023] In this specification, numerical ranges are provided for certain quantities. It should be understood that these ranges include all values ​​and subranges therein. Therefore, the range "from 50 to 80" includes all possible values ​​therein (e.g., 50, 51, 52, 53, 54, 55, 56, etc.) and all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). In addition, all values ​​in a given range can be the endpoints of the range covered by the range (e.g., the range 50-80 includes a range with endpoints, such as 55-80, 50-75, etc.).

[0024] The term "a / an" refers to one or more of the entity; for example, "a Polypeptide Conjugate" refers to one or more Polypeptide Conjugates or at least one Polypeptide Conjugate. Thus, the term "a / an" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein. In addition, the use of the indefinite article "a / an" to refer to "a Polypeptide Conjugate" does not exclude the possibility that more than one Polypeptide Conjugate is present, unless the context clearly requires that there is one and only one Polypeptide Conjugate.

[0025] As used herein, the term "adjacent" refers to two consecutive amino acids linked by a covalent bond. "Adjacent" is also used interchangeably with "continuous".

[0026] The term "carrier" refers to a compound, composition, substance or structure that, when combined with a compound or composition, aids or promotes the preparation, storage, administration, delivery, effectiveness, selectivity or any other characteristic of the compound or composition to achieve its intended use or purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and minimize any adverse side effects to the subject.

[0027] As used herein, "cell penetrating peptide" or "CPP" refers to any peptide, including proteins (ie, polypeptides), that can penetrate a cell membrane. As used herein, "cyclic cell penetrating peptide" or "cCPP" refers to any cyclic peptide that can penetrate a cell membrane.

[0028] As used herein, "foliar treatment" refers to a composition applied to the aerial parts or foliage of a plant or plant part, which may be leaves, stems, flowers, branches, or any aerial plant part, such as a scion.

[0029] As used herein, "linker" or "L" refers to a moiety that covalently links two or more components of a polypeptide conjugate disclosed herein (e.g., a linker can covalently link a CPP and a group that binds to a nucleic acid sequence through electrostatic interactions (i.e., P). In some embodiments, the linker can be a natural or non-natural amino acid or polypeptide. In other embodiments, the linker is a synthetic compound containing two or more suitable functional groups suitable for binding, for example, to a CPP and independently to P. In some embodiments, the linear length of the linker is about 3 to about 100 (e.g., about 3 to about 20) atoms (excluding branch atoms or substituents). In some embodiments, the distance between the linker and the two groups it links is about 1 Å to about 400 Å.

[0030] As used herein, "polypeptide" refers to at least two amino acid segments interconnected by peptide bonds. There is no upper limit to the number of amino acids contained in a polypeptide. In addition, a polypeptide may include non-natural amino acids, amino acid analogs, or other synthetic molecules that can be incorporated into a polypeptide.

[0031] As used herein, "monomer" refers to an amino acid residue in a polypeptide. In some embodiments, the amino acid monomer is divalent. In other embodiments, if the monomer is further substituted, the amino acid monomer may be trivalent. For example, a cysteine ​​monomer may independently form a peptide bond at the N and C termini, and may also form a disulfide bond.

[0032] As used herein, "amino acid analogs" or "analogs" (e.g., "arginine analogs," "lysine analogs," or "histidine analogs") refer to amino acid variants that retain at least one function of an amino acid, such as the ability to bind to an oligonucleotide via electrostatic interactions. Such variants may have extended or shortened side chains (e.g., one or more -CH 2 -group, it retains the ability of oligonucleotide binding by electrostatic interaction, or, modification can improve the ability of oligonucleotide binding by electrostatic interaction.For example, arginine analogs can include extra methylene or ethylene between main chain and guanidine / guanidinyl.Other examples include amino acids (for example, Me, Et, halogen, mercaptan, methoxyl group, ethoxyl group, C1-haloalkyl, C2-haloalkyl, amine, guanidine etc.) with one or more additional substituents.Amino acid analogs can be monovalent, divalent or trivalent.

[0033] In this specification, peptides and amino acid monomers are described as electrically neutral species. It should be understood that such species may be positively or negatively charged, depending on the conditions. For example, at pH 7, the N-terminus of an amino acid is protonated and positively charged (-NH 3 + ), while the C-terminus of the amino acid is deprotonated and negatively charged (-CO 2- ). Similarly, the side chains of some amino acids may be positively or negatively charged.

[0034] Each amino acid can be a natural amino acid or a non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a homologue of a natural amino acid because it has a structure similar to that of a natural amino acid, so it simulates the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and / or amino acid analogs that are not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Therefore, the term "amino acid" as used herein refers to natural and non-natural amino acids and analogs and derivatives thereof. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives or combinations thereof. Amino acid analogs encompass amino acids with structures similar but not identical to amino acids, for example due to modifications to the side chains or backbones of the amino acids. Such modifications can increase the hydrophobicity of the side chain, including extending the side chain by one or more hydrocarbons, or increasing the solvent accessible surface area of ​​amino acids with aromatic rings on the side chain (such as SASA described herein), for example by conjugating a second aromatic ring or increasing the size of the aromatic ring. Amino acid derivatives encompass natural and non-natural amino acids that have been modified (e.g., by substitution) to include a hydrophobic group as described herein. For example, derivatives of lysine include lysine whose side chain is substituted by an alkylamide group. These and other contents are listed in Table 1 together with the abbreviations used herein.

[0035] Table 1. Amino acid abbreviations

[0036]

[0037] *Single letter abbreviations: When shown in upper case letters herein, they represent the L-amino acid form, and when shown in lower case letters herein, they represent the D-amino acid form.

[0038] "Alkyl" or "alkyl group" refers to a fully saturated, straight or branched hydrocarbon chain radical having from one to twelve carbon atoms and attached to the rest of the molecule by a single bond. Alkyl groups containing any number of carbon atoms from 1 to 12 are included. Alkyl groups containing up to 12 carbon atoms are C 1 -C 12 Alkyl, containing up to 10 carbon atoms is C 1 -C 10 Alkyl, containing up to 6 carbon atoms is C1 -C 6 Alkyl, and the alkyl containing up to 5 carbon atoms is C 1 -C 5 Alkyl. C 1 -C 5 Alkyl groups include C 5 Alkyl, C 4 Alkyl, C 3 Alkyl, C 2 Alkyl and C 1 Alkyl (i.e. methyl). 1 -C 6 Alkyl groups include those mentioned above for C 1 -C 5 All parts of the alkyl group, but also including C 6 Alkyl. C 1 -C 10 Alkyl groups include those mentioned above for C 1 -C 5 Alkyl and C 1 -C 6 All parts of the alkyl group, but also including C 7 , C 8 , C 9 and C 10 Similarly, C 1 -C 12 Alkyl includes all of the aforementioned moieties, but also includes C 11 and C 12 Alkyl. C 1 -C 12 The non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl. Unless specifically stated otherwise in this specification, alkyl may be optionally substituted.

[0039] "Alkylene" or "alkylene chain" refers to a fully saturated, straight or branched, divalent hydrocarbon chain radical having from one to forty carbon atoms. 2 -C 40 Non-limiting examples of alkylene include ethylene, propylene, n-butylene, pentylene, and the like. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted as described herein.

[0040] "Alkenyl" or "alkenyl group" refers to a straight or branched hydrocarbon chain radical having from two to twelve carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl groups containing any number of carbon atoms from 2 to 12 are included. Alkenyl groups containing up to 12 carbon atoms are C 2 -C12 Alkenyl, alkenyl containing up to 10 carbon atoms is C 2 -C 10 Alkenyl, alkenyl containing up to 6 carbon atoms is C 2 -C 6 alkenyl, and alkenyl containing up to 5 carbon atoms is C 2 -C 5 Alkenyl. C 2 -C 5 Alkenyl includes C 5 Alkenyl, C 4 Alkenyl, C 3 Alkenyl and C 2 Alkenyl. C 2 -C 6 Alkenyl includes the above C 2 -C 5 All parts of alkenyl, but also including C 6 Alkenyl. C 2 -C 10 Alkenyl includes the above C 2 -C 5 Alkenyl and C 2 -C 6 All parts of alkenyl, but also including C 7 , C 8 , C 9 and C 10 Similarly, C 2 -C 12 Alkenyl includes all of the aforementioned moieties, but also includes C 11 and C 12 Alkenyl. C 2 -C 12Non-limiting examples of alkenyl groups include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl and 11-dodecenyl. Unless otherwise specifically stated in the specification, the alkyl group may be optionally substituted.

[0041] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain radical having 2 to 40 carbon atoms and having one or more carbon-carbon double bonds. 2 -C 40 Non-limiting examples of alkenylene include ethenylene (-CH=CH-), propenylene, butenylene, etc. Unless stated otherwise specifically in the specification, an alkenylene chain may be optionally substituted.

[0042] "Alkynyl" or "alkynyl group" refers to a straight or branched hydrocarbon chain radical having from 2 to 12 carbon atoms and having one or more carbon-carbon triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl groups containing any number of carbon atoms from 2 to 12 are included. Alkynyl groups containing up to 12 carbon atoms are C 2 -C 12 Alkynyl, containing up to 10 carbon atoms, is C 2 -C 10 Alkynyl, containing up to 6 carbon atoms, is C 2 -C 6 Alkynyl, and alkynyl containing up to 5 carbon atoms is C 2 -C 5 Alkynyl. C 2 -C 5 Alkynyl groups include C 5 Alkynyl, C4 Alkynyl, C 3 Alkynyl and C 2 Alkynyl. C 2 -C 6 Alkynyl groups include the above C 2 -C 5 All parts of the alkynyl group, but also including C 6 Alkynyl. C 2 -C 10 Alkynyl groups include the above C 2 -C 5 Alkynyl and C 2 -C 6 All parts of the alkynyl group, but also including C 7 , C 8 , C 9 and C 10 Similarly, C 2 -C 12 Alkynyl includes all of the aforementioned moieties, but also includes C 11 and C 12 Alkynyl. C 2 -C 12 Non-limiting examples of alkenyl include ethynyl, propynyl, butynyl, pentynyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted.

[0043] "Alkyne" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain radical having from 2 to 40 carbon atoms and having one or more carbon-carbon triple bonds. 2 -C 40 Non-limiting examples of alkynylene include ethynylene (-C≡C-), propargylene, etc. Unless stated otherwise specifically in the specification, an alkynylene chain may be optionally substituted.

[0044] "Aryl" refers to a hydrocarbon ring system comprising hydrogen, 6 to 40 carbon atoms and at least one aromatic ring. For purposes of the present disclosure, aryl can be a monovalent or divalent group (excluding substituents), which can be a monocyclic, bicyclic, tricyclic or tetracyclic system, and can include a condensed ring or bridged ring system. Aryl includes but is not limited to groups derived from acenaphthene, acenaphthene, phenanthrene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenanthrenes, phenanthrenes, pyrene and triphenylene. In some embodiments, when used as a joint or as a part of a joint, aryl can be divalent. Unless otherwise specifically stated in this specification, aryl can be optionally substituted.

[0045] As used herein, "aromatic" refers to an unsaturated cyclic molecule having 4n+2π electrons, where n is any integer. The term "non-aromatic" refers to any unsaturated cyclic molecule that does not fall within the definition of aromatic.

[0046] "Carbocyclyl", "carbocyclic ring" or "carbocycle" refers to a ring structure in which the atoms forming the ring are each carbon. The carbocycle may contain 3 to 20 carbon atoms in the ring. Carbocycles include aryl and cycloalkyl groups as well as fully unsaturated, partially unsaturated and fully saturated rings. In some embodiments, when used as a joint or part of a joint, the carbocyclyl may be divalent. Unless specifically stated otherwise in this specification, the carbocyclyl may be optionally substituted.

[0047] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon group having 3 to 40 carbon atoms and at least one ring, wherein the ring is composed only of carbon and hydrogen atoms, which may include a condensed ring or a bridged ring system. For the purposes of this disclosure, a cycloalkyl group may be a monovalent or divalent group (excluding substituents). Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl groups include, for example, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclo [2.2.1] heptyl, etc. In some embodiments, when used as a part of a joint or a joint, a cycloalkyl group may be divalent. Unless otherwise specifically stated in this specification, a cycloalkyl group may be optionally substituted.

[0048] "Cycloalkenyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 40 carbon atoms, at least one ring having and one or more carbon-carbon double bonds, wherein the ring is composed only of carbon and hydrogen atoms, which may include condensed rings or bridged ring systems. For purposes of the present invention, cycloalkenyl may be a monovalent or divalent group (excluding substituents). Monocyclic cycloalkenyls include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, etc. Polycyclic cycloalkenyls include, for example, bicyclo [2.2.1] hept-2-enyl, etc. In some embodiments, cycloalkenyl may be divalent when used as a part for a joint or a joint. Unless specifically stated otherwise in this specification, cycloalkenyl may be optionally substituted.

[0049] "Cycloalkynyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 40 carbon atoms, at least one ring and one or more carbon-carbon triple bonds, wherein the ring is composed only of carbon and hydrogen atoms, which may include a condensed ring or a bridged ring system. For purposes of the present invention, cycloalkynyl may be a monovalent or divalent group (excluding substituents). Monocyclic cycloalkynyl includes, for example, cycloheptynyl, cyclooctynyl, etc. In some embodiments, cycloalkynyl may be divalent when used as a part for a joint or a joint. Unless specifically stated otherwise in this specification, cycloalkynyl may be optionally substituted.

[0050] "Heterocyclyl", "heterocyclic ring" or "heterocycle" refers to a stable 3- to 20-membered aromatic ring group consisting of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. For the purposes of the present invention, a heterocyclyl group may be a monovalent or divalent group (excluding substituents). Heterocyclyl or heterocyclic ring includes heteroaryl groups as defined below. Unless otherwise expressly stated in this specification, a heterocyclyl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and may include a fused or bridged ring system; the nitrogen, carbon or sulfur atoms in the heterocyclyl group may be optionally oxidized; the nitrogen atom may be optionally quaternized; the heterocyclyl group may be partially saturated or fully saturated. Examples of such heterocyclic groups include, but are not limited to, dioxolanyl, thienyl [1,3] dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidone, pyrrolidinyl, pyrazolidinyl, quinuclidine, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl and 1,1-dioxothiomorpholinyl. In some embodiments, when used as a part of a joint or a joint, the heterocyclic group can be divalent. Unless otherwise specifically stated in this specification, the heterocyclic group can be optionally substituted.

[0051] "Heteroaryl" refers to a 5- to 20-membered ring system radical containing hydrogen atoms, 1 to 14 carbon atoms, 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of the present invention, a heteroaryl group can be a monovalent or divalent group (excluding substituents), can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, can include fused or bridged ring systems; the nitrogen, carbon or sulfur atoms in the heteroaryl group can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azaquinone, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl (benzothienyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, isothiazolyl, Imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidized pyridinyl, 1-oxidized pyrimidinyl, 1-oxidized pyrazinyl, 1-oxidized pyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl and thienyl (i.e., thienyl). In some embodiments, when used as a linker or part of a linker, the heteroaryl group can be divalent. Unless stated otherwise specifically in the specification, a heteroaryl group may be optionally substituted.

[0052] The term "ether" as used herein refers to a straight or branched divalent radical -[(CH 2 ) m -O-(CH 2 ) n ] z -, wherein m, n and z are each independently selected from 1 to 40. Examples include, but are not limited to, polyethylene glycol. Unless otherwise specifically stated in the specification, ethers may be optionally substituted.

[0053] As used herein, the term "substituted" means that at least one hydrogen atom in any of the above groups (i.e., alkylene, alkenylene, alkynylene, aryl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl and / or ether) is replaced by a bond to a non-hydrogen atom, such as, but not limited to: a halogen atom, e.g., F, Cl, Br and I; an oxygen atom in groups such as hydroxyl, alkoxy and ester groups; a sulfur atom in groups such as thiol, sulfanyl, sulfone, sulfonyl and sulfoxide groups; a group such as amine, amide, alkylamine, dialkylamine, arylamine, alkylarylamine, diarylamine, N- -Nitrides, imides, and enamines; silicon atoms in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl; and other heteroatoms in various other groups. "Substitution" also means that one or more hydrogen atoms in any of the above groups are replaced by higher bonds (e.g., double or triple bonds) to heteroatoms such as oxygen in oxo, carbonyl, carboxyl, and ester groups, and to nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substitution" includes any of the above groups in which one or more hydrogen atoms are replaced by -NR g R h 、-NR g C(=O)R h 、-NR g C(=O)NR g R h 、-NR g C(=O)OR h 、-NR g SO 2 R h 、-OC(=O)NR g R h 、-OR g 、-SR g 、-SOR g 、-SO 2 R g 、-OSO 2 R g 、-SO 2 OR g =NSO 2 R g and-SO 2 NR g R h Substitution also refers to the replacement of one or more hydrogen atoms in any of the above groups with -C(=O)R g 、-C(=O)OR g 、-C(=O)NR g R h 、-CH 2 SO 2 R g 、-CH2 SO 2 NR g R h In the above, R g and R h The same or different, and independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, sulfanyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. "Substitution" also refers to one or more hydrogen atoms in any of the above groups being replaced by a bond formed with amino, cyano, hydroxyl, imino, nitro, oxo, thio, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, sulfanyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. In addition, each of the above substituents may also be optionally substituted with one or more of the above substituents. In addition, one skilled in the art will recognize that "substituted" also encompasses the situation where one or more atoms on any of the above groups are replaced by a substituent listed in this paragraph, and the substituent forms a covalent bond with the CPP, P or L. For example, in certain embodiments, any of the above groups can be substituted at the first position with a carboxylic acid (i.e., -C(=O)OH) that forms an amide bond with lysine in the CPP, or can be substituted at the second position with a thiol group that forms a disulfide bond with cysteine ​​(or an amino acid analog having a thiol group).

[0054] As used in the specification and the claims that follow, a residue of a chemical substance refers to the resulting product of the chemical substance in a particular reaction scheme or subsequent formulation or a portion of a chemical product, whether or not that portion is actually derived from the chemical substance. Thus, an amino acid residue in a peptide or protein refers to one or more -OC(O)CH(R)NH- units in the peptide or protein.

[0055] The symbols used in this paper are " ” (hereinafter referred to as “connection point bond”) represents a connection point bond between two chemical entities, one of which is depicted as being connected to the connection point bond and the other chemical entity is not depicted as being connected to the connection point bond. For example, “ " indicates that the chemical entity "XY" is bound to another chemical entity through a point of attachment bond. In addition, specific points of attachment to undescribed chemical entities can be assigned by inference. For example, the compound CH 3 -R 3 , where R 3 H or " ”, it is inferred that when R 3 When it is "XY", connect the point key with R3 Connect to CH 3 The keys are the same.

[0056] Unless otherwise indicated, formulas with chemical bonds shown only as solid lines, rather than wedges or dashed lines, encompass every possible isomer, such as every enantiomer and diastereomer, and mixtures of isomers, such as racemic or non-racemic mixtures. The compounds described herein may contain one or more asymmetric centers, and thus may produce diastereomers and optical isomers. Unless otherwise indicated, the compounds and compositions disclosed herein include all of these possible diastereomers and their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers as well as isolated specific stereoisomers are also included. In the course of synthetic methods for preparing such compounds, or in the use of racemization or epimerization methods known to those skilled in the art, the product of such methods may be a mixture of stereoisomers.

[0057] Many organic compounds exist in optically active forms that can rotate the plane of plane polarized light. When describing optically active compounds, prefixes D and L or R and S are used to represent the absolute configuration of the molecule about its chiral center. The prefixes d and l or (+) and (-) are used to represent the rotation mark of the plane polarized light of the compound, wherein (-) or (-) are used to represent that the compound is left-handed. Compounds with (+) or d prefixes are right-handed. For a given chemical structure, these compounds called stereoisomers are identical, except that they are mirror images that cannot overlap each other. Specific stereoisomers can also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 enantiomeric mixture is referred to as a racemic mixture. Many compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, an asterisk (*) can be used to indicate chiral carbon. When the bond to a chiral carbon is described as a straight line in a disclosed formula, it is understood that both the (R) and (S) configurations of the chiral carbon, and therefore both enantiomers and mixtures thereof, are included in the formula. As used in the art, when it is necessary to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be described as a wedge (bond to an atom above the plane), while the other can be described as a series or wedge-shaped short parallel lines (bond to an atom below the plane). The Cahn-Inglod-Prelog system can be used to specify the (R) or (S) configuration as a chiral carbon.

[0058] The compounds described herein contain atoms in both their natural isotopic abundance and unnatural abundance. The disclosed compounds may be isotopically labeled or isotopically substituted compounds, which are identical to those compounds described, but for the fact that one or more of their atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that may be incorporated into the compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as respectively 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F and 36 Cl. Compounds further include prodrugs thereof, and pharmaceutically acceptable salts of the compounds or prodrugs containing the above isotopes and / or other isotopes of other atoms are within the scope of the present invention. Certain isotopically labeled compounds (e.g., radioactive isotopes such as 3 H and 14 C-incorporated) can be used in drug and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred because they are easy to prepare and detect. In addition, heavier isotopes such as deuterium (i.e., 2 H) Substitution may offer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and therefore may be preferred in some circumstances. Isotopically labeled compounds and prodrugs thereof can generally be prepared by carrying out the following procedures, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0059] Disclosed are components for preparing the compositions disclosed herein and the compositions themselves used in the methods disclosed herein. These and other materials are disclosed herein, and it should be understood that when the combinations, subsets, interactions, groups, etc. of these materials are disclosed, although the specific references of each different individual and common combination and arrangement of these compounds cannot be explicitly disclosed, each is specially considered and described herein. For example, if a specific compound is disclosed and discussed, then the discussion can be modified in a variety of ways to multiple molecules including the compound, with special consideration of each and every combination and arrangement of the compound and possible modifications, unless otherwise specifically indicated. Therefore, if a class of molecules A, B and C and a class of molecules D, E and F and an example of a disclosed combination molecule AD are disclosed, then even if there is no separate statement that each is a combination of meanings considered individually and collectively, it is also considered to disclose AE, AF, BD, BE, BF, CD, CE and CF. Similarly, any subset or these combinations are also disclosed. Therefore, for example, it will also be considered that the disclosed subgroups AE, BF and CE. This concept applies to all aspects of this patent application, including but not limited to steps in the method for preparing and using the compositions disclosed herein. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods disclosed herein.

[0060] As used herein, the term "contacting" refers to bringing a disclosed compound into contact with a target (e.g., a cell, target receptor, transcription factor, or other biological entity) in such a way that the compound can affect the activity of the target either directly; that is, through interaction with the target itself, or indirectly; that is, through interaction with another molecule, co-factor, factor, or protein on which the activity of the target depends.

[0061] As used herein, the terms "effective amount" and "amount effective" refer to an amount sufficient to achieve a desired result.

[0062] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

[0063] Compound

[0064] The present invention discloses cell-penetrating peptides and compositions containing them, which can provide universal vehicles for the cytoplasmic delivery of any peptide or protein cargo and other biomolecules including oligonucleotides. Compared with simple cell-penetrating peptides, the disclosed peptides can have higher cytosolic delivery efficiency and in vivo stability.

[0065] In a specific aspect, disclosed herein are peptides comprising: a membrane translocation domain having one or more cell penetrating peptide motifs, and a cargo portion linked to the membrane translocation domain, wherein the cargo portion comprises a plant bioactive portion, wherein at least one cell penetrating peptide motif is 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues.

[0066] Unlike the method of inserting a CPP motif into each target protein, the disclosed compounds, compositions and methods involve engineered membrane translocation domains that can be genetically or synthetically fused to any target cargo of interest. Another strategy disclosed herein is to split the CPP motif into two halves and insert them into two different regions of the membrane translocation domain, thereby greatly improving the cytoplasmic delivery efficiency.

[0067] In some embodiments, the compounds described herein can be used as plant activators. The term "plant activator" refers to a compound that activates the host plant's natural defense mechanisms such as systemic acquired resistance (SAR) or hypersensitivity reactions. The compositions can be used as plant activators for healthy plants and unhealthy plants, or plants in healthy and unhealthy environments.

[0068] In some embodiments, the compounds described herein can be used as plant stimulants. The term "plant stimulant" as used herein refers to a compound or composition applied to a plant under conditions of enhancing nutritional efficiency, stress tolerance and / or crop quality traits, regardless of its nutritional content. In particular, plant stimulants are used in the cultivation of plants to improve growth and development processes. The effect of stimulants on plants is not directly involved in the regulation of life processes, but rather affects metabolism in a broad sense. They can stimulate the synthesis of natural hormones, sometimes increase their activity, improve the absorption of minerals from the soil, and regulate the growth of roots. In addition, they can lead to increased resistance to adverse conditions (biological or abiotic). The use of stimulants in plant cultivation increases yields and also improves the quality of plants. Stimulants can enhance the life processes of plants without changing the natural behavior of plants. The compounds and / or compositions described herein can be plant stimulants, and therefore can be used as plant growth regulators, plant metabolic process regulators, plant physiological process regulators, substances that prevent plants from being affected by biological or abiotic stresses, and / or substances that provide plants with multiple disease resistance. The compositions are useful as plant stimulants on healthy and unhealthy plants, or plants in healthy and unhealthy environments.

[0069] Membrane translocation domain

[0070] The membrane translocation domain portion of the disclosed peptide can be any membrane translocation domain, i.e., a peptide sequence that can pass through a lipid bilayer, which has been modified to contain at least one cell penetration motif as described herein. In a preferred embodiment, there are two or three cell penetration motifs in the membrane translocation domain. For example, at least one cell penetrating peptide motif can be 3 to 10 amino acid residues in length and have at least three arginine and / or lysine residues, such as 4, 5 or 6 arginine and / or lysine residues. Alternatively, at least one cell penetrating peptide motif can be 3 to 10 amino acid residues in length and have at least two arginine and / or lysine residues, and at least one other cell penetrating peptide motif can be 2 to 8 amino acid residues in length and have at least two hydrophobic residues. When there are two or more cell penetrating peptide motifs, two or more arginine residues and / or lysine residues can be present within a span of 3 to 10 amino acids, and another cell penetrating peptide motif of two or more hydrophobic residues can be present within a span of 2 to 8 amino acids. The cell penetrating peptide motif can be located at any position of the membrane translocation domain.

[0071] In some embodiments, the membrane translocation domain can be a plant membrane translocation domain. In some instances, the membrane translocation domain can be a human membrane translocation domain, such as fibronectin type III. In a specific example, the membrane translocation domain has at least 90%, at least 95% or at least 97% sequence similarity to SEQ. ID. NO.: 118. In other examples, the membrane translocation domain is a human fibronectin type III with BC, DE, CD and FG loops, and the cell penetrating peptide motif is located in one or more of the BC, DE, CD or FG loops, for example, the cell penetrating peptide motif is located in two of the BC, DE, CD or FG loops, particularly in the BC and FG loops. These loops can be defined as having the following sequence BC = AVTVR (SEQ ID NO: 31); CD = GGNSPVQ (SEQ ID NO: 32); DE = PGSK (SEQ ID NO: 33); FG = GRGDSPAS (SEQ ID NO: 34).

[0072] In other examples, the membrane translocation domain can be any stably folded protein that can be efficiently expressed, preferably in bacteria. Some other examples of membrane translocation domains are nanobody scaffolds, DARPin scaffolds, and CTPR proteins (shared tetratricopeptide repeats; Acc. Chem. Res. 2021, 54, 4166−4177).

[0073] Cell penetrating peptide motif

[0074] The cell penetrating peptide (CPP) motif may comprise at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, or at least 6 amino acids, more specifically, 3 to 8, 3 to 6, 4 to 8, 4 to 6, or 6 to 8 amino acids. In most instances, the CPP motif is substituted into the membrane translocation domain such that the resulting peptide has the same number of amino acids as the native membrane translocation domain.

[0075] In some instances, at least two, three, four, five, six or seven amino acids of the CPP motif are adjacent arginine residues. In preferred instances, there are three, four or five adjacent arginine residues in the CPP motif. In other instances, the arginine residues are not adjacent in the CPP motif. Each amino acid in the CPP motif can be a natural or non-natural amino acid independently. When these adjacent arginine or lysine residues are CPP motifs, no additional CPP motifs are needed, such as CPP motifs with hydrophobic residues, although such hydrophobic CPP motifs can still be used. When the CPP motif contains two arginine residues, it is preferred that another CPP motif with at least two hydrophobic residues is present within 2 to 8 amino acids.

[0076] In other examples, at least one, at least two, at least three or more amino acids of the CPP motif are hydrophobic amino acids, i.e., have hydrophobic side chains. In some examples, the amino acids with hydrophobic side chains are independently selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylic acid, cyclohexylalanine, norleucine, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalene-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, tert-leucine or nicotinyllysine, each of which is optionally substituted with one or more substituents. In a specific example, each amino acid with a hydrophobic side chain is independently an amino acid with an aromatic side chain. In some embodiments, the amino acid with an aromatic side chain is 3-benzothienyl-L-alanine, naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan or tyrosine, each of which is optionally substituted with one or more substituents. Therefore, in some examples, the amino acid with a hydrophobic side chain is phenylalanine, naphthylalanine, tryptophan, or its analogs or derivatives naphthylalanine or tryptophan, or its analogs or derivatives. In other examples, the CPP motif also includes at least one phenylalanine, phenylglycine or histidine, or its analogs or derivatives.

[0077] , , , , , , .

[0078] In some examples, the CPP motif can include any combination of at least three adjacent arginines and at least two amino acids having hydrophobic side chains selected from aryl or heteroaryl, wherein the aryl and heteroaryl groups are optionally substituted, and the total number of amino acids in the CPP motif is in the range of 5 to about 8 amino acids.

[0079] In some examples, the membrane translocation domain is a human fibronectin type III having BC, DE, CD and FG loops, and the CPP is located in one or more of the BC, DE, CD or FG loops. For example, the CPP motif is located in two of the BC, DE, CD or FG loops. In a specific example, the CPP motif is located in the BC and DE, CD and FG loops, preferably in the BC and FG loops.

[0080] When two or more CPP motifs are present, one CPP motif may be a 3 to 10 amino acid segment having at least two arginine and / or lysine residues, and the other may be a 2 to 8 amino acid segment having at least two hydrophobic residues. For example, a membrane translocation domain may have two or more CPPs, and at least one motif is 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues.

[0081] In this example, the membrane translocation domain can be human fibronectin type III having BC, DE, CD and FG loops, and the CPP motif can be located in the BC loop and has 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, and the CPP motif can be located in the FG loop and has 3 to 10 amino acid residues and has at least three adjacent arginine and / or lysine residues. Alternatively, the CPP motif can be located in the FG loop and has 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, and the CPP motif can be located in the BC loop and has 3 to 10 amino acid residues and has at least three adjacent arginine and / or lysine residues.

[0082] When the CPP motif contains 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, it can be WW, FF, WF, FW, WWW, FFF, WFW, FWF, WWF, WFF, FWW, FFW, WYW, WWH, YWW or WYH. This CPP motif is preferably located in the BC loop. Further preferably, this CPP motif is WW, FW, WF, WYW, WWW, WWH, YWW, WYH or YWH.

[0083] A CPP motif having 3 to 10 amino acid residues and having at least three adjacent arginine and / or lysine residues may include RRR, RRRR, RRRRR. It may also be any combination of arginine and lysine residues. When this CPP motif is located in the FG loop, the length of I may be 3-10 residues and may be any combination of Arg and Lys (occasionally with other non-acidic residues). The CPP motif (e.g., WWWRRRR) may be selectively cleaved so that some Arg / Lys residues are moved from the FG loop to the BC loop (e.g., WWWR…RRR, WWWRR…RR, WWWRRRR…etc.); the CPP motif (e.g., WWWRRRR) may be selectively cleaved so that some hydrophobic residues are moved from the BC loop to the FG loop (e.g., WW…WRRR, W…WWRRRR, …WWWRRRR, etc.). The CPP motif (e.g., WWWRRRR) can be selectively cleaved such that the BC or FG loop contains a combination of hydrophobic and positively charged residues (e.g., WWR...WRRR, WWRR...WRR, WWRR...RRW, RRW...WWRR, etc.).

[0084] In specific examples, the CPP motif comprises SEQ. ID. NO: 104, 105, 11, 112, 113, 114, 115, 116 or 117.

[0085] In some examples, the CPP motif can be or include any sequence listed in Table 2. In some examples, the cell penetrating peptide can be or include the reverse sequence of any sequence listed in Table 2.

[0086] Table 2. CPP motif sequences

[0087]

[0088]

[0089]

[0090]

[0091] Φ = L-naphthylalanine; ϕ = D-naphthylalanine; Ω = L-norleucine; r = D-arginine; F = L-phenylalanine; f = D-phenylalanine; q = D-glutamine; Χ = L-4-fluorophenylalanine; Dap = L-2,3-diaminopropionic acid; Sar, sarcosine; F 2 Pmp, L-difluorophosphonomethylphenylalanine; Dod, dodecanoyl; Pra, L-propargylglycine; AzK, L-6-azido-2-aminohexanoic acid; Agp, L-2-amino-3-guanidinopropionic acid;b Cyclization between Pim and Nlys; c Cyclization between Lys and Glu; d Macrocyclization via multicomponent reaction with aziridine aldehydes and isonitriles; e Cyclization between the main chains of Gln residues; f The N-terminal amine and the side chains of the two Dap residues are dicyclized with Tm; g The three Cys side chains are bicyclized with tri(bromomethyl)benzene; h Pra and Azk were cyclized via a click reaction.

[0092] The chirality of amino acids can be selected to improve cytosolic absorption efficiency. In some embodiments, at least two amino acids have opposite chirality. In some embodiments, at least two amino acids with opposite chirality can be adjacent to each other. In some embodiments, at least three amino acids have alternating stereochemistry relative to each other. In some embodiments, at least three amino acids with alternating chirality relative to each other can be adjacent to each other. In some embodiments, at least two amino acids have the same chirality. In some embodiments, at least two amino acids with the same chirality can be adjacent to each other. In some embodiments, at least two amino acids have the same chirality and at least two amino acids have opposite chirality. In some embodiments, at least two amino acids with opposite chirality can be adjacent to at least two amino acids with the same chirality. Therefore, in some embodiments, the adjacent amino acids in cCPP can have any of the following sequences: DL, LD, DLLD, LDDL, LDLLD, DLDDL, DLLDL or LDDLD.

[0093] Cargo section

[0094] The cargo moiety can be attached to the membrane translocation domain.The cargo moiety can be attached to an amino group (eg, N-terminus), a carboxylic acid group (eg, C-terminus), or the side chain of one or more amino acids in the membrane translocation domain.

[0095] When the cargo moiety is attached to the amino acid side chain in the membrane translocation domain, the membrane translocation domain includes an amino acid having a side chain with a suitable functional group to form a covalent bond (conjugation) with the cargo, or can be modified to provide a suitable functional group (e.g., conjugated via a linker) to form a covalent bond with the cargo. In some embodiments, the amino acid on the membrane translocation domain with a side chain suitable for conjugation with the cargo is a cysteine ​​residue, a glutamic acid residue, an aspartic acid residue, a lysine residue, or a 2,3-diaminopropionic acid residue. In such an embodiment, the cargo can be directly conjugated to the side chain of the amino acid (e.g., by forming a disulfide bond with a cysteine ​​residue or an amide bond with a glutamic acid residue or a 2,3-diaminopropionic acid residue) or the cargo can be conjugated to the amino acid side chain via a linker (e.g., PEG).

[0096] In some embodiments, the cargo portion may include a plant bioactive portion. In some embodiments, the cargo portion may also include any cargo of interest, such as a linker portion, a detectable portion, or any combination thereof. In some examples, the cargo portion may include one or more additional amino acids (e.g., K, UK, TRV); a linker (e.g., a bifunctional linker LC-SMCC); coenzyme A; phospho-coumarylaminopropionic acid (pCAP); 8-amino-3,6-dioxaoctanoic acid (miniPEG); L-2,3-diaminopropionic acid (Dap or J); L-β-naphthylalanine; L-piperidinic acid (Pip); sarcosine; trimesic acid (Tm); 7-amino-4-methylcoumarin (Amc); fluorescein isothiocyanate (FITC); L-2-naphthylalanine; norleucine; 2-aminobutyric acid; rhodamine B (Rho); dexamethasone (DEX); or a combination thereof.

[0097] Bioactive parts of plants

[0098] The cargo portion can include a plant biologically active portion. In some embodiments, the detectable portion can be linked to the plant biologically active portion. The plant biologically active portion can be attached to the cell penetrating peptide portion at an amino group, a carboxylate group, or a side chain of any amino acid of the cell penetrating peptide portion (e.g., at an amino group, a carboxylate group, or a side chain or any amino acid of a CPP). In some examples, the plant biologically active portion can be attached to a detectable portion.

[0099] The term "plant bioactive fraction" refers to compounds that are capable of activating the host plant's natural defense mechanisms, such as systemic acquired resistance (SAR) or hypersensitive responses, or that are capable of enhancing nutrient efficiency, stress tolerance and / or crop quality traits, regardless of their nutritional content.

[0100] Non-limiting examples of plant biologically active portions can include, but are not limited to, synthetically derived or naturally occurring flagellin and flagellin-related polypeptides (including those conserved in Bacillus), thionin, harpin protein or polypeptide or harpin-like polypeptide, elongation factor Tu (EF-Tu), phytosulfuronidase (PSKα), root hair promoting polypeptide (RHPP), hypersensitive response elicitor protein or polypeptide, antifungal peptides, insect toxin peptides, antifreeze proteins, heat-resistant proteins, drought-resistant proteins, vitamin biosynthetic enzymes, bioherbicide peptides, or maize mitochondrial mutant proteins, or any combination thereof. Suitable synthetically derived or naturally occurring flagellin and flagellin-related polypeptides (including those conserved in Bacillus), thionine, harpin protein or polypeptide or harpin-like polypeptide, elongation factor Tu (EF-Tu), plant sulfopeptide (PSKα), root hair promoting polypeptide (RHPP) and / or hypersensitive response elicitor protein or polypeptides described herein or in International Application Publication Nos. WO 2019 / 018768, WO 2010 / 019442, WO 1998 / 054214, WO 2001 / 098501 and WO 2013 / 102189, each of which is incorporated herein by reference in its entirety. Plant bioactive parts can be selected according to their different modes of action and can be used alone or in combination with other polypeptides to adapt to specific agricultural needs. They can replace or supplement commercially available agrochemicals, biostimulants, supplementary bioactive substances, insecticidal compounds or any combination thereof.

[0101] Flagellins and flagellin-related peptides derived from these flagellins have been reported to play functional roles mainly in the innate immune response of plants. These peptides are derived from highly conserved domains of eubacterial flagellins. Flagellins are the main building blocks of bacterial flagella. Flagellin subunits that constitute bacterial flagellar filaments can act as potent inducers in cells to elicit defense-related responses in various plant species.

[0102] "Flagellin" is a globular protein that is arranged in a hollow cylinder to form the filaments in bacterial flagella. Flagellin is the major alternative to bacterial flagella and is found in flagellated bacteria. Plants can sense, resist infection and send out defense signals against bacterial microorganisms by recognizing conserved epitopes such as the 22 amino acid sequence (Flg22) located at the N-terminus of the full-length flagellin coding sequence. The inducible activity of the Flg22 polypeptide has been attributed to this conserved domain within the N-terminus of flagellin (Felix et al., 1999). Plants can sense bacterial flagellin through pattern recognition receptors (PRRs) on the surface of plant cells called flagellin-sensitive receptors, which are leucine-rich repeat receptor kinases located on the plasma membrane and available on the surface of plant cells. In plants, the best characterized PRR is FLAGELLIN SENSING 2 (FLS2), which is highly conserved in both monocots and dicots.

[0103] Plant defensins are also described as antimicrobial peptides (AMPs). Plant defensins contain several conserved cysteinyl residues that form disulfide bridges and contribute to their structural stability. Defensins are among the best characterized cysteine-rich AMPs in plants. Members of the defensin family have four disulfide bridges that fold into a globular structure. This highly conserved structure has a highly specialized role in protecting plants from microbial pathogens (Nawrot et al., “Plant antimicrobial peptides,” Folia Microbiology 59: 181-196, 2014).

[0104] Thionine is a plant AMP rich in cystine, belonging to the defensin family, usually containing 45-48 amino acid residues, of which 6-8 amino acids are cysteine, forming 3-4 disulfide bonds in higher plants. Thionine has been found to exist in monocots and dicots, and its expression can be induced by infection with various microorganisms (Tam et al., "Antimicrobial peptides from plants," Pharmaceuticals 8: 711-757, 2015). It has been found that certain specific amino acids in thionine (such as Lys1 and Tyr13) are highly conserved and are essential for the functional toxicity of AMPs.

[0105] Harpins and harpin-like proteins are similar to flagellin or flagellin-related polypeptides. Harpins include a group of bacterial inducers from larger precursor proteins. When harpins penetrate into the intercellular space or apoplast of plant cells, they are essential for triggering the hypersensitive response (HR) (Kim et al., "Mutational analysis of Xanthomonas harpinHpaG identifies a key functional region that elicits the hypersensitiveresponse in nonhost plants," Journal of Bacteriology 186: 6239-6247, 2004). Applying long-range harpin-like bioactive elicitor polypeptides to plants may provide another way to protect plants from disease and insect stress. Harpins utilize a type III secretion system that enables proteins to pass through the lipid bilayer that constitutes the plasma membrane of plants. Binding of harpin to the plasma cell membrane surface can trigger an innate immune response that is similar to that triggered by pathogen-associated molecular patterns (PAMPs) and is known to activate PAMP-triggered immunity (Engelhardt et al., “Separable roles of the Pseudomonas syringae pv. Phaseolicola accessory protein HrpZ1 inion-conducting pore formation and activation of plant immunity,” The Plant Journal 57: 706-717, 2009). Mutational analysis of harpin-like HpaG-derived polypeptides revealed that 12 amino acid residues between Leu-39 and Leu50 of the initial 133 amino acid harpin inducer precursor protein are critical for initiating hypersensitivity (HR) and subsequent innate immune responses in tobacco (Kim et al., “Mutational analysis of Xanthomonas harpin HpaG identifies a key functional region that elicits the hypersensitive response in nonhost plants,” Journal of Bacteriology 186:6239-6247, 2004). This suggests that a specific amino acid region of harpin, similar to other AMPs, is responsible for initiating the response.Harpins, such as HpaG-like proteins, can be used to enhance plant resistance not only to pathogens, but also to insects (Choi et al., "Harpins, multifunctional proteins secreted by gram-negative plantpathogenic bacteria," Molecular Plant Microbe Interactions 26: 1115-1122, 2013). Harpins have been used to induce disease resistance in plants and protect plants from infection and feeding by phloem-feeding insects (e.g., aphids) (Zhang et al., "Harpin-induced expression and transgenic overexpression of phloem protein gene At.PP2A1 in Arabidopsis repress phloemfeeding of the green peach aphid Myzus persicae," BMC Plant Biology 11: 1-11, 2011).

[0106] In some embodiments, harpin proteins or polypeptides or harpin-like polypeptides may include, but are not limited to, homologs of Erwinia amylovora HrpN, including homologs from species of Erwinia, Pantoea, and Firmicutes. Examples of such homologs include those harpin proteins identified in: Genbank Accession Nos. AAC31644 (Erwinia amylovora); AAQ21220, AAQ 17045, CAE25423, CAE25424, CAE25425 and CAF74881 (Erwinia amylovora); CAC20124, Q47278, Q47279 and AAY17519 (Erwinia chrysanthemi); CAE25422 (Erwinia chrysanthemi strain JP557); AAG01466 (Pantoea stutzeri); AAF76342 (Pantoea agglomerans); ABZ05760, ABI15988, ABI15989, ABI15990, ABI15991 991, ABI15992, ABI15996, ABK80762, ABD04037, ABI15994, ABD04035, ABD04036, AAY17521, AAX38231, ABI15995, AAQ73910 and CAL69276 (Pectobacterium carotovora); YP_050198, AAS20361 and CAE45180 (Pectobacterium black rot); and ABD22989 (Pectobacterium betavasculorum); each of which is incorporated herein by reference in its entirety.

[0107] Another group of harpin proteins or polypeptides or harpin-like polypeptides may include, but are not limited to, homologs of Erwinia amylovora HrpW and Pseudomonas syringae HrpW, including those from the genera Erwinia, Pseudomonas, Xanthomonas, Acidovorax, and Firmicutes.Examples of such homologs include those harpin proteins identified in Genbank Accession Nos. U94513, CAA74158, AAC04849, and AAF63402 (Erwinia amylovora); AAQ17046 (Ernonia amylovora); YP 00 1906489 (Erwinia tasmanica); YP_050207 (Pectobacterium nigrofurans); AF037983 (Pseudomonas syringae pv. tomato); AAO50075 (Pseudomonas syringae pv. bean); AAL84244 (Pseudomonas syringae pv. maculatum); AAX58537, AAX58557, AAX58525, AAX58531, AAX58527, AAX58577, AAX58491, AAX58515, ... AX58517, AAX58523, AAX58583, AAX58451, AAX58561, AAX58453, AAX58541, AAX58589, AAT96311, AAX58497, A AX58579, AAX58449, AAX58485, AAX58563, AAX58581, AAX58575, AAX58569, AAX58567, AAX58505, AAX58591, A AX58503, AAX58507, AAX58509, AAX58469, AAX58441, AAX58543, AAX58495, AAX58549, AAX58593, AAX58511, A AX58519, AAT96270, AAX58447, AAX58571, AAX58465, AAX58489, AAX58533, AAX58535, AAX58461, AAT96350, A AX58473, AAX58483, AAX58475, AAX58457, AAX52461, AAX52457, AAT96222 (Pseudomonas viridis); ABA47299 and BAG24117 (Pseudomonas cichorii); CAH57075 (Pseudomonas haliflavus); BAE80274 and BAE80242 (Acidovorax avenae); and AAM37767 (Xanthomonas tricholoma pv. citri); each of which is incorporated herein by reference in its entirety.

[0108] Another group of harpin proteins or polypeptides or harpin-like polypeptides can include, but are not limited to, homologs of Pseudomonas syringae HrpZ, including those from other species of the genus Pseudomonas. Examples of such homologs include those harpin proteins identified as follows: Genbank Accession Nos. P35674, AAB00127, ABL01505, AAQ92359, BAD20880, BAD20876, BAD20892, BAD20884, BAD20928, BAD20936, BAD20932, BAD20924, BAD20856, BAD20864, ​​BAD20860, BAD20848, BAD20844, BAD20836, BAD20840, BAD20824, BAD20842, BAD20820, BAD20916, BAD20872, BAC81526, and BAC81527. , BAA74798, BAD20904, AAB86735, BAD20912, BAD20908, ABL01504, BAB40655, ABO26225, ABO26228 (Pseudomonas syringae pv.); BAD20868 (Pseudomonas figi); AAX52452, AAT96159, AAX52266, AAX52396, AAT96322, AAT96281, AAX52272, AAX52306, AAX52270, AAX52402, AAX52276, AAX52318, AAX52262, and AAT96361 (Pseudomonas viridis); CAJ76697 (Pseudomonas haliflorata); YP OOl 185537 (Pseudomonas mendocina); and ABA47309 and BAG24128 (Pseudomonas cichorii); each of which is incorporated herein by reference in its entirety. Another group of harpin proteins or polypeptides or harpin-like polypeptides may include, but are not limited to, homologs of Xanthomonas campestris HreX (see U.S. Pat. No. 6,960,705 to Wei et al., which is incorporated herein by reference in its entirety), including those from other Xanthomonas species. Examples of such homologs include the harpin proteins identified in Genbank Accession Nos. NP_636614, YP_001904470, YP_362171 (Xanthomonas campestris); ABB72197, ABK51585, ABU48601, ABK51584, YPJ98734, and ZP_02245223 (Xanthomonas oryzae); and ABK51588 and NP_640771 (Xanthomonas tricholoma); each of which is incorporated herein by reference in its entirety.

[0109] In some embodiments, the harpin protein or polypeptide or harpin-like polypeptide is a fragment or a combination of fragments (i.e., a fusion protein) of one of the above-mentioned harpin proteins. In some embodiments, the harpin fragment or fusion protein includes a fragment that triggers a hypersensitivity reaction. In another embodiment, the harpin fragment or fusion protein includes a fragment that does not trigger a hypersensitivity reaction. Suitable harpin fragments include, for example, two structural units: a stable α-helical unit having a length of 12 or more amino acids; and a hydrophilic acidic unit having a length of 12 or more amino acids, which can be a β form, a β turn, or a disordered form. The fragment can also be characterized by an acidic pi value of preferably about 5 or less. The fragment can contain any number of amino acids, for example, about 25 to about 60 amino acids, or about 28 to about 40 amino acids.

[0110] Examples of suitable harpin fragments or fusion proteins are described in U.S. Patent No. 6,583,107 to Laby et al. and PCT Publication No. WO 01 / 098501 to Fan et al., each of which is incorporated herein by reference in its entirety. PCT Publication No. WO 01 / 098501 to Fan et al. also describes methods for obtaining fragments of harpin proteins or polypeptides that can be used in the present invention.

[0111] A harpin fragment or fusion protein, currently available from Plant Health Care Inc., is characterized by the following amino acid sequence of SEQ ID NO: 129:

[0112] MSLNTSGLGASTMQISIGGAGGNNGLLGTHMPGTSSSPGLFQSGGDNGLGGHNANSALGQQPIDRQTIEQMAQLLAELLKSLLDSGEKLGDNFGASADSASG TGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDSGPAKDGNANAGANDPSKNDPSKSQGPQSANKTGNVDDANNQDPMQALMQLLEDLVKLLKAALHMQQ PGGNDKGNGVGGDSGQNDDSTSGTDSTSDSSDPMQQLLKMFSEIMQSLFGDEQDGTDSTSGSRFTRTGIGMKAGIQALNDIGTHSDSSTRSFVNKGDRAMAKE IGQFMDQYPEVFGKPQYQKGPGQEVKTDDKSWAKALSKPDDDGMTPASMEQFNKAKGMIKSAMAGDTGNGNLQARGAGGSSLGIDAMMAGDAINMALGKLGAA

[0113] The harpin fragment or fusion protein of SEQ ID NO: 129 is encoded by the following nucleotide sequence of SEQ ID NO: 130:

[0114]

[0115] Elongation factor Tu is an abundant protein found in bacteria that can act as a pathogen-associated molecular pattern (PAMP) to initiate signaling cascades involved in plant disease resistance and plant innate immunity to microbial pathogens. Interestingly, some EF-Tu polypeptides have also been found in plants. The first 18 amino acid residues at the N-terminus of EF-Tu in Escherichia coli are called elf18, which is known to be a potent inducer of PAMP-induced immune responses in plants (Zipfel et al., "Perception of the bacterial PAMP EF-Tu by the Receptor EFR restricts Agrobacterium-mediated transformation," Cell 125: 749-760, 2006). Peptides derived from Escherichia coli EF-Tu can be sensed by the plant cell surface localization receptor EF-Tu receptor (EFR) (Zipfel et al., 2006). EF-Tu binding and EFR activation follow a similar mode of action as the Flg peptide-FLS2 receptor complex (Mbengue et al., “Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases,” Proc Natl Acad Sci USA 113: 11034-9, 2016).

[0116] Phytosulfokines (PSKs) belong to a class of sulfated plant peptides encoded by precursor genes that are ubiquitous and highly conserved in higher plants (Sauter M., “Phytosulfokine peptide signaling,” Journal of Experimental Biology 66: 1-9, 2015). PSK genes are encoded by a small gene family present in monocots and dicots, and encode PSK polypeptides that can function as pentapeptides or C-terminal truncated tetrapeptides (Lorbiecke R, Sauter M, “Comparative analysis of PSK peptide growth factor precursor homologs,” Plant Science 163: 348-357, 2002).

[0117] Phytosulfonyltransferase proteins target the secretory pathway of plants through conserved signal peptides (Lorbiecke R, Sauter M, "Comparative analysis of PSK peptide growth factor precursor homologs," Plant Science 163: 348-357, 2002). Phytosulfonyltransferases (PSKs) act as sulfated growth factors with biostimulatory activity, participating in the control of root and shoot apical meristem development, growth regulation, and reproductive processes. PSK has also been reported to initiate cell proliferation, differentiation of quiescent tissues, and to participate in the formation, stimulation, and differentiation of tubular elements (Matsubayashi et al., “The endogenous sulfated pentapeptide phytosulfokine-αstimulates tracheary element differentiation of isolated mesophyll cells of zinnia, Plant Physiology 120: 1043-1048, 1999). PSK signaling has also been reported to be involved in regulating the elongation of roots and hypocotyls in Arabidopsis seedlings (Kutschmar et al., “PSK-α promotes root growth in Arabidopsis,” New Phytologist 181: 820-831, 2009).

[0118] Root Hair Promoting Polypeptide (RHPP) is a 12 amino acid fragment derived from soybean Kunitz trypsin inhibitor (KTI) protein, which is synthesized from HA from Bacillus circulans. 12 RHPP was detected in soybean meal that was degraded by alkaline proteases (Matsumiya Y. and Kubo M. "Soybean and Nutrition, Chapter 11: Soybean Peptide: Novel plant growth promoting peptide from soybean," Agricultural and Biological Sciences, Sheny HE (ed.), pgs. 215-230, 2011). When applied to soybean roots, RHPP was shown to accumulate in the roots and promote root growth by stimulating cell division and root hair differentiation in Brassica.

[0119] Detectable part

[0120] The detectable portion can include any detectable label. Examples of suitable detectable labels include, but are not limited to, UV-Vis labels, near-infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photocleavage moieties, chelating centers, heavy atoms, radioisotopes, isotope detectable spin resonance labels, paramagnetic moieties, chromophores, or any combination thereof. In some embodiments, the label can be detected without the addition of other reagents.

[0121] In some embodiments, the detectable moiety is a biocompatible detectable moiety, so that the compound can be suitable for a variety of biological applications. "Biocompatible" as used herein generally refers to such compounds and any metabolites or degradation products thereof that are generally non-toxic to cells and tissues, and do not cause any significant adverse effects to cells and tissues when cells and tissues are incubated (e.g., cultured) in the presence of such compounds.

[0122] The detectable moiety may comprise a luminophore, such as a fluorescent label or a near infrared label. Examples of suitable luminophores include, but are not limited to, metalloporphyrins; benzoporphyrins; azabenzoporphyrins; naphthoporphyrins; phthalocyanines; polycyclic aromatic hydrocarbons, such as perylene, perylene diimide, pyrene; azo dyes; xanthene dyes; boron dipyrromethene, azaboron dipyrromethene, cyanine dyes, metal ligand complexes such as bipyridine, bipyridyls, phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium; acridine, oxazine derivatives such as benzophenoxazine; azaannulene, squaryl cyanine; 8-hydroxyquinoline, polymethine, luminescent nanoparticles, such as quantum dots, nanocrystals; quinolones; terbium complexes; inorganic phosphors; ion carriers such as crown ethers attached or derived dyes; or combinations thereof. Specific examples of suitable luminophores include, but are not limited to, palladium(II) octaethylporphyrin; platinum(II)-octaethylporphyrin; palladium(II) tetraphenylporphyrin; platinum(II) tetraphenylporphyrin; palladium(II) meso-tetraphenylporphyrin tetrabenzoporphyrin; platinum(II) meso-tetraphenylmethylbenzoporphyrin; palladium(II) octaethylporphyrinone; platinum(II) octaethylporphyrinone; palladium(II) meso-tetra(pentafluorophenyl)porphyrin; platinum(II) meso-tetra(pentafluorophenyl)porphyrin; ruthenium(II) tris(4,7-diphenyl-1,10-phenanthroline) (Ru(dpp) 3 ); Ruthenium(II) tris(1,10-phenanthroline)(Ru(phen) 3 ), tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate (Ru(bpy) 3); erythrosine B; fluorescein; fluorescein isothiocyanate (FITC); eosin; iridium (III) ((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)); indium (III) ((benzothiazol-2-yl)-7-(diethylamino)-coumarin))-2-(acetylacetonate); Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow; Texas Red; Rhodamine B; Rhodamine 6G; Sulforhodamine; m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue; bromocresol green; bromocresol red; bromothymol blue; Cy2; Cy3; Cy5; Cy5.5; Cy7; 4-nitrophenol; alizarin; phenolphthalein; o-cresolphthalein; Chlorophenol red; calcium magnesium apatite; bromoxylenol; phenol red; neutral red; nitrazine; 3,4,5,6-tetrabromophenolphthalein; Congo red; fluorescein; eosin; 2',7'-dichlorofluorescein; 5(6)-carboxyfluorescein; carboxynaphthofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid; semi-naphtho-rhodamine fluorescein; semi-naphthofluorescein; tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)dichloride; (4,7-diphenyl-1,10-phenanthroline)ruthenium(II)tetraphenylboron; platinum(II) octaethylporphyrin; dialkylcarbocyanine; dioctadecylepoxycarbocyanine; fluorenylmethoxycarbonyl chloride; 7-amino-4-methylcoumarin (Amc); green fluorescent protein (GFP); and derivatives or combinations thereof.

[0123] In some examples, the detectable moiety can include rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), naphthofluorescein (NF), or a derivative or combination thereof.

[0124] The detectable moiety can be attached to the cell penetrating peptide portion at an amino group, a carboxylate group, or a side chain of any amino acid of the cell penetrating peptide portion (eg, at an amino group, a carboxylate group, or a side chain or any amino acid of a CPP).

[0125] Connectors

[0126] In various embodiments, the joint is covalently bound to the amino acid on the membrane translocation domain. The joint can be any part of the membrane translocation domain and the cargo part. In some embodiments, the joint can be an amino acid. In other embodiments, the precursor of the joint can be any suitable molecule, which can form two or more bonds with the amino acid in the membrane translocation domain and the cargo part. Therefore, in various embodiments, the precursor of the joint has two or more functional groups, and each functional group can form a covalent bond with the membrane translocation domain and the cargo part. For example, the joint can be covalently bound to the N-terminus, C-terminus or side chain of any amino acid in the membrane translocation domain or a combination thereof. In specific embodiments, the joint forms a covalent bond between the membrane translocation domain and the cargo part. In some embodiments, the joint can be an unstructured polypeptide sequence. In some embodiments, when the joint is an unstructured polypeptide sequence, it allows the membrane translocation domain, the joint and the cargo conjugate to be recombinantly produced.

[0127] In some embodiments, the linker is selected from the group consisting of at least one amino acid, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ether, each of which can be optionally substituted as defined above. Non-limiting examples of linkers include polyethylene glycol, optionally conjugated to a lysine residue.

[0128] In some embodiments, the linker length can be 0 to 1000 amino acids. In some embodiments, the linker can be designed as a Gly-Gly-Ser repeating sequence. In some embodiments, the linker sequence can be a linker described in Adv Drug Deliv Rev. 2013 Oct 15;65(10): 1357–1369. For example, (G) n (n=1-10) or SEQ IDNO: 131(GGGGS) n (n=1-4) defined flexible linker sequence, such as SEQ ID NO: 131 GGGGS, SEQ ID NO: 132 (GGGGS) 3 ; From SEQ ID NO: 133A (EAAAK) n A(n=2-5) or (XP) n (n=5-20) defined rigid linker sequence, wherein X represents any amino acid, preferably Ala, Lys or Glu, such as SEQ ID NO: 134A (EAAAK) 4 ALEA(EAAAK)4 A , SEQ ID NO: 135 AEAAAKEAAAKA, SEQ ID NO: 136 PAPAP, (Ala-Pro) n(n=5-17); cleavable linker sequence, such as disulfide bond, protease sensitive sequence, (e.g., SEQ ID NO: 137 VSQTSKLTR↓AETVFPDV b 、SEQ ID NO: 138 PLG↓LWA c , SEQ ID NO: 139 RVL↓AEA, SEQ ID NO: 140 EDVVCC↓SMSY, SEQ ID NO: 141 GGIEGR↓GS c , SEQ ID NO: 142 TRHRQPR↓GWE, SEQ ID NO: 143AGNRVRR↓SVG, SEQ ID NO: 144 RRRRRRR↓R↓R d or SEQ ID NO: 145 GFLG↓ e ,in a Protease-sensitive cleavage sites are indicated by “↓”; b Factor XIa / FVIIa-sensitive cleavage; c Matrix metalloproteinase-1 sensitive cleavage sequences, examples of which are provided herein; d HIV PR (HIV-1 protease); NS3 protease (HCV protease); Xa factor-sensitive cleavage, respectively; e Furin-sensitive cleavage; and f Cathepsin B-sensitive cleavage).

[0129] In some embodiments, the linker is covalently bound to the N or C terminus of an amino acid on a CPP motif, or to a side chain of glutamine, asparagine or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). In specific embodiments, the linker forms a bond with the glutamine side chain on the CPP motif. In other specific embodiments, the linker described herein has the structure of L-1 or L-2:

[0130] ,

[0131] in

[0132] AA s It is the side chain or end of an amino acid on a peptide or stapler;

[0133] AA c It is the side chain or end of the amino acid of cCPP;

[0134] p is an integer from 0 to 10; and

[0135] q is an integer from 1 to 50.

[0136] In some embodiments, the linker is capable of releasing the cargo moiety from the membrane translocation domain after the polypeptide conjugate enters the cytoplasm of the cell. In some embodiments, the linker contains a group, or forms a group after binding to the membrane translocation domain and the cargo moiety, which is cleaved after the polypeptide conjugate is taken up by the cytosol, thereby releasing the cargo moiety. Non-limiting examples of physiologically cleavable linking groups include carbonates, thiocarbonates, thioethers, thioesters, disulfides, sulfoxides, hydrazines, protease-cleavable dipeptide linkers, and the like.

[0137] For example, in an embodiment, the linker is covalently bound to the membrane translocation domain via a disulfide bond, e.g., wherein the side chain of a cysteine ​​or cysteine ​​analog is located in the membrane translocation domain or the cargo portion. In some embodiments, the disulfide bond is formed between a thiol group on the linker precursor and a side chain of a cysteine ​​or amino acid analog having a thiol group on the peptide, wherein the bond to hydrogen on each thiol group is replaced by a bond to a sulfur atom. Non-limiting examples of amino acid analogs having thiol groups that can be used with the Polypeptide Conjugates disclosed herein are discussed above.

[0138] Preparation method

[0139] The compounds described herein can be prepared in various ways known to those skilled in the art of organic synthesis or variations thereof as understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions can vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0140] The modification of compound as described herein includes the addition, removal or movement of various components (constituent) as described for each compound. Similarly, when there are one or more chiral centers in a molecule, the chirality of the molecule can be changed. In addition, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection and the selection of appropriate protecting groups can be determined by those skilled in the art. The chemical property (chemistry) of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, the 4th edition, Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0141] The starting materials and reagents used in preparing the disclosed compounds and compositions can be purchased from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or by methods known to those skilled in the art according to procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplements (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials such as the pharmaceutical carriers disclosed herein can be obtained from commercial sources.

[0142] The reactions used to produce the compounds described herein can be carried out in a solvent, which can be selected by a person skilled in the art of organic synthesis. The solvent may be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) under which the reactions are carried out. The reactions can be carried out in one solvent or in a mixture of more than one solvent. The formation of products or intermediates can be monitored according to any suitable method known in the art. For example, the formation of products or intermediates can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (such as high performance liquid chromatography (HPLC) or thin layer chromatography) to monitor product formation.

[0143] The disclosed compounds can be prepared by expression and purification like any other protein. See Chen, K. and Pei, D. (2020). Engineering Cell-Permeable Proteins through Insertion of Cell-Penetrating Motifs into Surface Loops. ACS chemical biology, 15(9), 2568–2576, which is incorporated herein by reference in its entirety to teach methods for preparing proteins. Other methods for preparing the disclosed compositions involve solid phase peptide synthesis, in which the amino acid α-N-terminus is protected by an acid or base protecting group. Such a protecting group should have stable properties under peptide bond formation conditions while being easily removed without destroying the growing peptide chain or racemizing any chiral center contained therein. Suitable protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, tert-pentyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfinyl, 2-cyano-tert-butyloxycarbonyl, etc. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. For side chain amino groups such as lysine and arginine, other preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc and adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopentyl and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for aspartic acid and glutamic acid, benzyl and t-butyl; for cysteine, triphenylmethyl (trityl). In solid phase peptide synthesis, the α-C terminal amino acid is attached to a suitable solid support or resin. Solid supports suitable for the above synthesis are those materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction and are insoluble in the medium used. The solid support used for the synthesis of the α-C-terminal carboxyl peptide is 4-hydroxymethylphenoxymethyl-co-poly(styrene-1% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin, available from Applied Biosystems (Foster City, Calif.).The α-C-terminal amino acid is coupled to the resin by N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) or o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), with or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris(dimethylamino)phosphine hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), at a temperature between 10° C. and 50° C. in a solvent such as dichloromethane or DMF for about 1 to about 24 hours. When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine (preferably piperidine) prior to coupling with the above α-C-terminal amino acid. One method of coupling with deprotected 4 (2', 4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin is to combine o-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) in DMF. The coupling of consecutive protected amino acids can be carried out in an automatic peptide synthesizer. In one example, the α-N terminus of the amino acid of the growing peptide chain is protected by Fmoc. Removal of the Fmoc protecting group from the α-N-terminus of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced at approximately 3-fold molar excess, and the coupling is preferably carried out in DMF.

[0144] The coupling agents can be o-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, which can be performed continuously or all at once. The removal and deprotection of the polypeptide can be completed in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thianisole, water, ethanedithiol and trifluoroacetic acid. In the case where the α-C terminus of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification (e.g., with methanol) followed by aminolysis or direct transamidation. At this point, the protected peptide can be purified or directly proceed to the next step. Removal of side chain protecting groups can be accomplished using the above-mentioned cleavage mixture. The fully deprotected peptide can be purified by a series of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; partition chromatography, for example on Sephadex G-25, LH-20, or countercurrent partition; high performance liquid chromatography (HPLC), particularly reversed phase HPLC on octyl or octadecyl silica bonded phase column packings.

[0145] How to use

[0146] Provided herein are methods of using the compounds and / or compositions described herein. Also provided are methods of delivering a plant stimulant into a plant cell, comprising contacting the plant cell with a peptide described herein. Also provided are methods of delivering a plant activator into a plant cell, comprising contacting the plant cell with a peptide described herein. Also provided are methods of delivering a plant bioactive portion into a plant cell, comprising contacting the plant cell with a peptide described herein.

[0147] Also provided are methods of delivering plant stimulants to plants, comprising contacting the plants with the peptides described herein. Also provided are methods of delivering plant activators to plants, comprising contacting the plants with the peptides described herein. Also provided are methods of delivering plant bioactive parts to plants, comprising contacting the plants with the peptides described herein.

[0148] Also described are methods of treating plants suffering from a disease caused by a pathogen. The methods may include contacting the plant with an effective amount of a compound and / or composition described herein.

[0149] In some aspects, the compounds or compositions described herein can be used to protect plants from biotic stress caused by living organisms (e.g., fungi, bacteria, nematodes, insects, mites, and animals); stimulate seeds during germination; protect plants from abiotic stress caused by physical or chemical stress sources of abiotic origin (e.g., the presence of harmful chemicals (including salts), limited water supply, sunburn, frost damage, wind damage, nutrient deficiencies, or improper cultural practices (e.g., overwatering or planting too deep)); enhance plant growth, yield, health, longevity, productivity, and / or vigor; and / or provide plants with multiple disease resistance.

[0150] Also provided are methods of protecting plants from biotic stress; stimulating seeds during germination; protecting plants from abiotic stress; enhancing plant growth, yield, health, lifespan, productivity and / or vigor; providing plants with multiple disease resistance; or any combination thereof. The method may comprise contacting a plant cell with a peptide described herein.

[0151] Compounds or compositions described herein provide resistance of plants to multiple pathogens. In some aspects, compounds or compositions can be used as plant stimulants for plants suffering from diseases caused by pathogens. Pathogens can include fungi, viruses, bacteria, mycoplasmas, spiroplasmas, or viroids. Exemplary pathogens can include fungi, such as Erisyphe polyni, Phytophthora capsicci, Verticillium dahliae, and other Verticillium spp, powdery mildew, and Fusarium; bacteria, such as Pseudomonas syringae pv. tomato, and viruses, such as tobacco mosaic virus and brome mosaic virus. Other exemplary pathogens include: Colletotrichum lagenarum, Pyricularia oryzae, Pseudomonas lachrymans, Xanthomonas oryzae, Xanthomonas vesicatoria, Phytophthora infestans ontomatoes, Plasmopara viticola, Pseudomonas tomato, Phytophthora parasitica var. nicotiniae, Peronospora tabacina, Cercospora nicotianae, Pseudomonas tabaci, Erysiphe graminis, Phytophthora alfalfa, Phytophthora medicaginis), Phytophthora macrosporum (P.megasperma), rice blast (Pyricularia oryzae), leaf blight (such as Helminthosporium oryzae), rice false smut (Cochliobolus miyabeanus), leggy growth (such as Gibberella fujikuroi), seedling blight (such as Rhizopus oryzae), sheath blight (such as Rhizoctonia solani), crown rust (Puccinia coronata), powdery mildew (such as Erysiphe graminis), powdery mildew (such as Rhynchsporium secalis), grain smut (Cochliobolus sativus), grain black spot (Helminthosporium gramineum), grain black spot (Pyrenophora gramineum), grain black spot (Pyrenophora teres), Tilletia caries, Ustilago nuda, Leptosphaeria nodorum, Septoria nodorum, Puccinia striiformis, Typhula incarnata, Pseudocercosporella herpotrichoides, Calonectria graminicola, Fusarium nivale, Puccinia graminis, Typhula ishikariensis, Puccinia recondita, Puccinia triticina, Helminthosporium gramineum, Ustilago tritici, Pythium debaryanum, Fusarium nivale, Puccinia graminis, Typhula ishikariensis, Puccinia recondita, Puccinia triticina, Helminthosporium gramineum, Ustilago tritici, Pythium debaryanum, Fusarium nivale), Phytophthora infestans, Peronospora tabacina, Phytophthora parasitica var.), mosaic disease, Pythium debaryanum, Rhizoctonia solani, Pythium aphanidermatum, Botrytis cinerea, Botrytis cinerea, Mycosphaerella arachidicola, Rosellinia nectrix, Alternaria leaf spot spot), and other diseases of cereals, grain crops, sugar beets, legumes, pome fruits, stone fruits, fruits, citrus fruits, oil crops, cucumber plants, fiber crops, lauraceae, ornamental plants and vegetables, such as rapeseed, sunflower, carrot, pepper, strawberry, melon, kiwi, onion, scallion, sweet potato, fig, plum, asparagus, persimmon, soybean, red bean, watermelon, chrysanthemum, spinach, lettuce, asparagus, cabbage, carrot, onion, tomato, potato, bell pepper, tea, wheat, barley, rye, oatmeal, etc. Wheat, rice, sorghum, sugar beets, fodder beets, apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries, beans, lentils, peas, soybeans, rapeseed, mustard, poppies, olives, sunflowers, coconuts, castor beans, cocoa beans, peanuts, cucumbers, zucchini, melons, cotton, flax, hemp, hemp, citrus, lemons, grapefruit, oranges, avocados, cinnamon, camphor, corn, tobacco, nuts, coffee, sugar cane, tea, grapevines, hops, bananas, natural rubber plants, flowers, shrubs, deciduous and coniferous trees, etc.

[0152] Compounds or compositions described herein are effective against a variety of insects. European corn borer is a major pest of corn (corn and sweet corn), but also feeds on more than 200 plants, including green beans, wax beans, lima beans, soybeans, peppers, potatoes, tomatoes, and a variety of weeds. Other insect pests that damage a variety of vegetable crops and feed on insect larvae include, but are not limited to, beet armyworm, cabbage looper, corn earworm, fall armyworm, diamondback moth, cabbage root maggot, onion maggot, corn seed maggot, beet worm (melon worm), pepper maggot, and tomato pinworm.

[0153] About using these compounds or compositions to enhance plant growth, various forms of plant growth enhancement or promotion can be realized. This may occur when plants start growing from seeds, and may also occur in the later stage of plant life. For example, plant growth according to the present invention encompasses higher yield, increased seed yield, increased seed germination percentage, increased plant size, larger biomass, more and larger fruit, earlier fruit coloring and earlier fruit and plant maturation. For example, early germination and early maturity allow crops to grow in regions with short growing seasons, otherwise these regions can not grow. The raising of seed germination percentage improves the growing manner of crops and improves the utilization efficiency of seeds. Higher yield, larger area and stronger biomass yield make it possible to produce more income from given land. In some embodiments, compound or composition can be used for promoting early flowering.

[0154] As used herein, "health of a plant" or "plant health" refers to the condition of a plant and / or its products, which is determined by several aspects alone or in combination with each other, such as increased yield, plant vigor, quality and tolerance to abiotic and / or biotic stresses.

[0155] Plants that are attacked by fungi or pesticides generally produce less biomass, which results in reduced yields compared to plants that have been treated with therapeutic or preventive treatments for pathogenic fungi or any other relevant pests and can be grown without damage from biotic stress factors. However, even in the absence of any biotic stress, the application of the compounds and / or compositions described herein also enhances the health of the plants. The application of the compounds and / or compositions described herein to plants and / or areas of cultivation can also be carried out in the absence of pest pressure on the plants.

[0156] According to the present invention, "increasing plant yield" means that the yield of a plant product is increased by a measurable amount compared to the yield of the same plant product produced under the same conditions but without the application of the compound and / or composition to the plant and / or cultivation area. In one embodiment, the term "yield" refers to fruits in the appropriate sense, as well as vegetables, nuts, grains and seeds.

[0157] “Grains” and “fruits” are to be understood as any plant product which can be further utilized after harvest, such as true fruits, vegetables, nuts, grains, seeds, wood (e.g. in the case of forestry plants), flowers (e.g. in the case of horticultural and ornamental plants), etc.

[0158] Increased plant yield can be characterized by the following non-limiting characteristics: increased plant weight; increased biomass, such as higher total fresh weight (FW) and / or higher total dry weight (DW); increased number of flowers per plant; higher grain and / or fruit yield; more tillers or lateral shoots (branches); larger leaves; increased shoot growth; increased protein content; increased oil content; increased starch content; increased pigment content; increased chlorophyll content; and any combination thereof.

[0159] Chlorophyll content is positively correlated with the photosynthesis rate of a plant, so the higher the chlorophyll content, the higher the plant's yield.

[0160] Increasing plant yield may involve increasing plant vigor. Plant vigor is manifested in several aspects, including the overall visual appearance of the plant. Improved plant vigor may be characterized by, inter alia: improved plant vitality; improved plant growth; improved plant development; improved visual appearance; improved plant stand (less plant leaning / lodging); improved seedling emergence; enhanced root growth and / or a more developed root system; enhanced nodulation, particularly rhizobium nodulation; larger leaves; larger size; increased plant height; increased number of tillers; increased number of lateral branches; increased number of flowers per plant; increased shoot growth; increased root growth (extensive root system); enhanced photosynthetic activity; enhanced pigment content; earlier flowering; earlier fruiting; earlier and better germination; earlier grain maturity; fewer unproductive tillers; fewer dead basal leaves; fewer required inputs (e.g., fertilizer or water); greener leaves; full maturity with a shortened growing cycle; less fertilizer required; less seed sowing required; easier harvesting; faster and more uniform maturity; longer shelf life; longer panicles; delayed senescence; stronger and / or more productive tillers; better extractability of ingredients; improved seed quality (for sowing for seed production in subsequent seasons); reduced production of ethylene and / or inhibition of plant uptake of ethylene; and any combination thereof.

[0161] The enhancement of plant photosynthetic activity can be based on the increase of plant stomatal conductance and / or CO 2 Increased absorption rate.

[0162] Increasing the yield of a plant may involve improving the quality of the plant and / or its products. Improvements in plant quality may include, but are not limited to, improving certain plant characteristics, such as increasing the content and / or composition of certain ingredients by a measurable or significant amount compared to the same plant factors produced under the same conditions, but without applying the composition of the present invention. The enhanced quality may be characterized in particular by increased nutritional ingredients; increased protein content; increased fatty acid content; increased metabolite content; increased carotenoid content; increased sugar content; increased amount of essential amino acids; improved nutritional composition; improved protein composition; improved fatty acid composition; improved metabolite composition; improved carotenoid composition; improved sugar composition; improved amino acid composition; improved or optimal fruit color; improved leaf color; higher storage capacity; higher processability of harvested products; or any combination thereof.

[0163] Increasing the yield of a plant may involve increasing the tolerance or resistance of the plant to biotic and / or abiotic stress factors. Biological and abiotic stresses, particularly long-term stresses, can have deleterious effects on plants. Biological stresses are caused by living organisms, while abiotic stresses are caused by, for example, extreme environments. In one embodiment, applying the compounds and / or compositions described herein to plants according to the methods of the present invention enhances tolerance or resistance to biotic and / or abiotic stress factors, meaning that: (1) certain negative factors caused by biotic and / or abiotic stresses are reduced in measurable or perceptible amounts compared to plants exposed to the same conditions but not treated with the compounds and / or compositions described herein, and (2) the negative factors are not reduced by a direct effect of the composition on the stress factor, such as by its fungicidal or insecticidal action that directly destroys microorganisms or pests, but by stimulating the plant's own defense response to the stress factor.

[0164] Negative factors caused by biotic stresses such as pathogens and pests are well known and range from foliage wilting to complete destruction of plants.Biotic stresses can be caused by living organisms such as pests (e.g., insects, spiders, and nematodes), competing plants (e.g., weeds), microorganisms (e.g., plant pathogenic fungi and / or bacteria), and / or viruses.

[0165] Negative factors caused by abiotic stress are also well known and can usually be observed as reduced plant vigor (as described above) or the following symptoms: leaf spotting, leaf "burning", reduced growth, reduced flowering, reduced biomass, reduced crop yield, reduced crop nutritional value and delayed crop maturity, to name a few. Abiotic stress may be caused by, among other factors, extreme temperatures, such as heat or cold (heat stress / cold stress), drastic changes in temperature, abnormal temperatures in a particular season, drought (drought stress), extreme humidity, high salinity (salt stress), radiation (e.g. increased UV radiation due to the decline of the ozone layer), elevated ozone levels (ozone stress), organic pollution (e.g. caused by phytotoxic pesticides), inorganic pollution (e.g. caused by heavy metal pollutants) and any combination thereof.

[0166] Biotic and / or abiotic stress factors reduce the quantity and quality of stressed plants, crops and fruits. In terms of quality, reproductive development may be affected, affecting crops where fruits or seeds are important. Protein synthesis, accumulation and storage are mainly affected by temperature; almost all types of stress slow down growth; polysaccharide synthesis (structural and storage) is reduced or altered. These effects lead to a decrease in biomass (yield) and changes in the nutritional value of plant products.

[0167] The plant health indicators identified above may be interdependent and may also influence each other. For example, an increased resistance to biotic and / or abiotic stresses may lead to a more vigorous plant, for example producing better and larger crops, thus increasing yields. Conversely, a more developed root system may enhance resistance to biotic and / or abiotic stresses.

[0168] Application of the compounds and / or compositions described herein to plants and / or areas under cultivation can produce a synergistic effect on plants to: increase plant health, increase plant yield, increase plant biomass, increase plant oil content, increase plant vigor, increase plant stand, increase plant emergence, increase plant root growth, increase plant photosynthetic activity, improve plant quality, improve plant nutritional composition, improve plant protein composition, improve plant carotenoid composition, increase plant tolerance to biotic stress, increase plant tolerance to fungi, increase plant tolerance to nematodes, increase plant tolerance to bacteria, increase plant tolerance to abiotic stress, increase plant tolerance to drought stress, increase plant tolerance to cold stress, increase plant tolerance to heat stress, increase plant tolerance to salt stress, increase plant tolerance to ozone stress, and / or any combination thereof.

[0169] One of the most important factors in enhancing resistance to biotic and abiotic stresses is the stimulation of the plant's natural defense responses, which is achieved by applying the compounds and / or compositions described herein according to the methods described herein.

[0170] Any known technique for applying botanicals can be used to contact the plant (including its roots, flowers, leaves or stems) with the disclosed compounds or compositions.

[0171] Exemplary application technology includes but is not limited to spraying, atomizing, dusting, spreading, sprinkling, drip irrigation, dipping, soaking, injection, hydroponics or directly applying in water (in water). In some embodiments, suitable composition can include the composition for HV, LV and ULV spraying and the composition for ULV cold spray and warm spray preparation. Application method may be different because of the intended purpose. Composition can be applied to the plant in the field or greenhouse. In some respects, composition can be applied to the part of plant before planting, for example, applied on tuber. In some embodiments, compound or composition can be applied on plant surface or plant plasma membrane as foliar spray.

[0172] The composition can be contacted with any part of the plant, such as the root or leaves of the plant. In some embodiments, the composition can be contacted with the root by spraying the soil, mechanical incorporation, mixing with fertilizers, soil amendment, premixing or the like.

[0173] In some embodiments, composition can be contacted with plant seeds. The seed treatment agent containing compound or composition can use any commercially available seed treatment machinery to apply, or can also use any acceptable non-commercial method to apply, such as using syringe or any other seed treatment device. The general seed treatment coating program using compound or composition can be carried out using Wintersteiger HEGE 11 (Wintersteiger AG, Austria, Germany), and applied to the seed of major crops (i.e. corn, soybean, wheat, rice and various vegetables).

[0174] Seeds can be coated by a variety of methods, including but not limited to pouring or pumping, showering or spraying an aqueous solution containing a compound or composition on the seeds, spraying or applying to the seed layer with or without a delivery system. Suitable mixing devices include drums, mixing bowls or rollers, or other fluid application devices including a bowl or drum for holding seeds during coating.

[0175] Compositions described herein can be contacted with plants intermittently. In some aspects, plants can be contacted with the composition twice or more. For example, plants can be contacted with the composition 3, 4, 5, 6, 7, 8, 9 or 10 times. In some embodiments, plants can be contacted with the composition 2 to about 5 times. In some embodiments, plants can be contacted with the composition once. In some aspects, plants can be contacted with the composition once every 5 to 21 days. For example, plants can be contacted with the composition once every 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. In some embodiments, plants can be contacted with the composition once a week. In some aspects, plants can be contacted with the composition 1 to 5 times every 5 to 21 days. For example, plants can be contacted about 1 to about 5 times per week.

[0176] In some aspects, the compositions described herein can be applied prior to the onset of a stress factor.

[0177] The term "plant" as used herein includes the whole plant and its parts, including but not limited to the vegetative organs / structures of the stem (e.g., leaves, stems and tubers), roots, flowers and flower organs / structures (e.g., bracts, sepals, petals, stamens, pistils, anthers and ovules), seeds (including embryos, endosperms and seed coats) and fruits (mature ovaries), plant tissues (e.g., vascular tissues, ground tissues, etc.) and cells (e.g., guard cells, egg cells, etc.) and their progeny. "Plant cell" as used herein refers to any plant cell, and may include cells on the surface of the plant or inside the plant plasma membrane, such as epidermal cells, trichome cells, xylem cells, phloem cells, sieve tube elements or companion cells.

[0178] Plant classes useful in the methods described herein include both higher and lower plant classes, including angiosperms (monocots and dicots), gymnosperms, ferns, horsetails, gymnophytes, lycophytes, bryophytes, and multicellular algae. For example, plants useful in the methods described herein include any vascular plant, such as a monocot, dicot, or gymnosperm, including, but not limited to, alfalfa, apple, Arabidopsis, banana, barley, rapeseed, castor bean, chrysanthemum, clover, cocoa, coffee, cotton, cottonseed, corn, crambe, cranberry, cucumber, dendrobium, cassava, eucalyptus, fescue, flax, gladiolus, lily, linseed, millet, melon, mustard, oat, oil palm, rapeseed, papaya, peanut, pineapple, ornamental plants, yew, potato, rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, sugar beet, sugar cane, sunflower, strawberry, tobacco, tomato, turf grass, wheat and vegetable crops such as lettuce, celery, broccoli, cauliflower, cucurbits, onions (including garlic, shallots, leeks and chives); fruit and nut trees such as apples, pears, peaches, oranges, grapefruits, lemons, limes, almonds, pecans, walnuts, hazelnuts; vines such as grapes, kiwis, hops; fruit shrubs and thorns such as raspberries, blackberries, gooseberries; forest trees such as ash, pine, fir, maple, oak, chestnut, poplar; preferably alfalfa, rapeseed, castor bean, corn, cotton, sea kale, flax, linseed, mustard, oil palm, rapeseed, peanuts, potatoes, rice, safflower, sesame, soybeans, sugar beets, sunflowers, tobacco, tomatoes and wheat. In some embodiments, plants used in the methods described herein include any crop plants, such as forage crops, oilseed crops, cereal crops, fruit crops, vegetable crops, fiber crops, spice crops, nut crops, turf crops, sugar crops, beverage crops, and forestry crops.

[0179] Plants can be divided into agricultural plants, forestry plants, ornamental plants and horticultural plants according to their use and / or consumption by humans. In addition, "plants" include natural or wild plants, as well as genetically modified plants.

[0180] "Agricultural" plants are plants that are harvested or grown, in whole or in part, on a commercial scale, or that are important sources of feed, food, fiber (e.g., cotton and flax), fuel (e.g., wood, bioethanol, biodiesel, and biomass), or other chemical compounds. Agricultural plants also include vegetables. Thus, crops include cereals (e.g., wheat, rye, barley, triticale, oats, sorghum, and rice); beets (e.g., sugar beets or fodder beets); legumes (e.g., beans, lentils, peas, alfalfa, and soybeans); oilseed plants (e.g., rapeseed, rapeseed, mustard, Brassica juncea); juncea), linseed, mustard, olives, sunflowers, cocoa beans, castor oil plants, oil palm, peanuts and soybeans); cucurbits (e.g., pumpkin, cucumber and melon); fiber plants (e.g., cotton, flax, hemp and jute); vegetables (e.g., cucumbers, spinach, lettuce, asparagus, cabbage, carrots, radishes, turnips, celery, endive, chicory, Brussels sprouts, parsnips, cauliflower, broccoli, garlic, eggplant, peppers, squash, onions, tomatoes, potatoes, sweet potatoes, cucurbits and paprika); laurels (e.g., avocados, cinnamon and camphor); energy and feedstock plants (e.g., corn, soybeans, rapeseed, canola, sugar cane and oil palm); tobacco; nuts (including peanuts); coffee; tea; vines (e.g., table grapes and juice vines); hops; stone fruits; apples; blueberries; strawberries; pears; citrus; raspberries; pineapples; sugar cane; turf, natural rubber plants and hemp.

[0181] "Garden plants" are plants commonly used in gardening, including but not limited to ornamental plants, vegetables and fruits. "Ornamental" plants are plants commonly used in gardening, such as in parks, gardens, balconies and terraces. Non-limiting examples of ornamental plants include turf, geranium, pelargonia, petunia, begonia and fuchsia. Non-limiting examples of vegetables are as described above. Non-limiting examples of fruits include apples, pears, cherries, strawberries, citrus, peaches, apricots and blueberries.

[0182] "Forestry" plants are understood to be trees, more specifically trees used for reforestation or industrial plantations. Industrial plantations are usually used for the commercial production of forest products, such as timber, pulp, paper, rubber trees, Christmas trees or young trees for gardening. Non-limiting examples of forestry plants are conifers (e.g., pine), in particular fir and spruce of the genus Pinus; eucalyptus; tropical species (e.g., teak, rubber tree, oil palm); willow (Salix), in particular willow; poplar (Cottonwood), in particular poplar; beech, in particular Fagus; birch; oil palm; cherry, walnut and oak.

[0183] As mentioned above, the term "plant" also includes plants improved from wild types. This change can be achieved by breeding, mutagenesis or genetic engineering (including transgenic and non-transgenic plants). Plants modified by genetic engineering include plants whose genetic material is modified using recombinant DNA technology. This modification generally includes modifications that are not easily obtained by hybridization, mutation or natural recombination under natural conditions. Generally, one or more genes are integrated into the genetic material of genetically modified plants to improve certain characteristics of plants. Examples of genetically modified plants include, but are not limited to, crops that tolerate the effects of herbicides, fungicides or pesticides through breeding including genetic engineering methods, or plants with improved characteristics compared to existing plants, which can be produced by, for example, traditional breeding methods and / or the production of mutants or by recombinant procedures. Examples of genetically modified plants also include those that, by using recombinant DNA technology, are able to synthesize one or more proteins to increase the resistance or tolerance of these plants to bacterial, viral or fungal pathogens; plants that increase the productivity of these plants (e.g., biomass production, grain yield, starch content, oil content and / or protein content), tolerance to drought, salinity or other growth-limiting environmental factors or tolerance to pests and fungal, bacterial or viral pathogens, and / or plants containing altered amounts of substances or new contents of substances, in particular to improve raw material production and / or to improve human or animal nutrition, such as potatoes that produce increased amounts of amylopectin (e.g., oil crops that produce health-promoting long-chain ω-3 fatty acids or unsaturated ω-9 fatty acids (e.g., Nexera® rapeseed, DOW Agro Sciences, Canada), Amflora® potato, BASF SE, Germany).

[0184] The methods for producing such genetically modified plants are generally known to the person skilled in the art and are described, for example, in the above-mentioned references.

[0185] Compositions, formulations and methods of administration

[0186] Also disclosed herein are compositions comprising the compounds described herein.

[0187] Agricultural formulations of active substances are well known. Non-limiting examples include solids, semisolids, liquids, solutions, suspensions, emulsions, gels, oil dispersions, capsules (e.g., active ingredients are encapsulated in microcapsules), pulvis, powders, pastes, particles, etc. The specific formulation selected may vary according to a specific mode of application. In each case, it is generally advantageous to ensure that the active ingredient is fine and uniformly distributed in a liquid or solid carrier. The composition described herein can be in any suitable form according to its intended use. In some aspects, the composition can be in the form of an aqueous solution. In some aspects, the composition can be a solution comprising an organic solvent such as an alcohol. In some aspects, the composition can be a solution comprising an organic solvent and an inorganic solvent mixture.

[0188] Preferably, the compositions described herein are formulated in a manner suitable for large or small scale agricultural and horticultural applications.

[0189] The selected dosage level of the composition will depend on a variety of factors, including, for example, the route of administration, the time of administration, the duration of treatment, other drugs and / or materials used in conjunction with the specific compound used, the condition and overall health of the plant being treated, and similar factors well known in the agricultural field. However, the compositions described herein can provide plant stimulation even at low doses. One of ordinary skill in the art can readily determine and prescribe the effective amount of the desired composition.

[0190] Methods of formulation are taught, for example, in U.S. Pat. No. 3,060,084 to Littler and European Patent No. 0707445 to BASF AG (for liquid concentrates); Browning, "Agglomeration," Chemical Engineering pp. 147-48 (1967); Perry's Chemical Engineer's Handbook, 4th ed., McGraw-Hill, New York, 1963; PCT Publication No. WO 2000-0106 to E.I. Du Pont De Nemours and Co. No. 91 / 13546; U.S. Patent No. 4,172,714 to Albert; U.S. Patent No. 4,144,050 to Frensch et al.; U.S. Patent No. 3,920,442 to Albert; U.S. Patent No. 5,180,587 to Moore; U.S. Patent No. 5,232,701 to Ogawa et al.; U.S. Patent No. 5,208,030, British Patent No. 2,095,558 to Hoy et al.; U.S. Patent No. 3,299,566 to Macmullen; Klingman, Weed Control as a Science, J. Wiley & Sons, New York, 1961; Hance et al., Weed Control Handbook, 8th Edition, Blackwell Scientific, Oxford, 1989; and Mollet and Grubemann, Formulation Technology, Wiley VCH Verlag, Weinheim, 2001, each of which is incorporated herein by reference in its entirety.

[0191] As mentioned above, the composition can include an agriculturally effective amount of a combination of a compound described herein and an agriculturally acceptable carrier, and can also include other adjuvants. The composition can be formulated (together or alone) in a manner commonly used in agricultural chemical formulations. For example, the composition can include adjuvants commonly used in agricultural chemical formulations. The specific adjuvants used can depend on the specific application form and active substance, respectively. Non-limiting examples of suitable adjuvants include carriers, adjuvants, diluents, thickeners, buffers, preservatives, surfactants, wetting agents, coating agents, monosaccharides, polysaccharides, abrasives, insecticides, pesticides, herbicides, nematicides, bactericides, fungicides, acaricides, fertilizers, biostimulants, colorants, humectants, antifreeze agents, defoamers, compatibilizers, chelating agents, neutralizers and buffers, corrosion inhibitors, osmotic protectants, odorants, antibiotics, spreading agents, dispersants, freezing point depressants, antimicrobial agents, crop oils, safeners, adhesives, protective colloids, emulsifiers, tackifiers, amino acids, biological control agents or combinations thereof.

[0192] In some embodiments, the compositions described herein may include pesticides, including insecticides, herbicides, fungicides, bactericides, nematicides, miticides, or any combination thereof. The pesticides may be applied to the plants simultaneously or sequentially. In some embodiments, the pesticides are applied to the plants after applying the compounds and / or compositions described herein.

[0193] In some aspects, the composition can be used in addition to water, wetting agents, tackifiers, dispersants or emulsifiers to prepare a composition concentrate suitable for dilution.

[0194] Agriculturally acceptable carriers may include organic carriers or inorganic carriers. Exemplary carriers include, but are not limited to, water, organic solvents, inorganic solvents, petroleum fractions or hydrocarbons (such as mineral oil), aromatic solvents, paraffin oil, vegetable oils (such as soybean oil, rapeseed oil, olive oil, castor oil, sunflower oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil), esters of the above vegetable oils, esters of monohydric alcohols or dihydric, trihydric or other low polyols (containing 4-6 hydroxyl groups) (such as 2-ethylhexyl stearate, n-butyl oleate, isopropyl myristate , propylene glycol dioleate, dioctyl succinate, dibutyl adipate, dioctyl phthalate), esters of mono-, di- and polycarboxylic acids, toluene, xylene, naphtha, crop oils, acetone, methyl ethyl ketone, cyclohexanone, trichloroethylene, perchloroethylene, ethyl acetate, amyl acetate, butyl acetate, propylene glycol monomethyl ether and diethylene glycol monomethyl ether, methanol, ethanol, isopropanol, amyl alcohol, ethylene glycol, propylene glycol, glycerol, N-methyl-2-pyrrolidone, N,N-dimethylalkylamides, dimethyl sulfoxide, liquid fertilizers and mixtures thereof. Other exemplary carriers include silica, silica gel, silicates, talc, kaolin, limestone, lime, chalk, laterite, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, pyrophyllite clay, attapulgite clay, diatomaceous earth, calcium carbonate, bentonite, Fuller's earth, cottonseed hulls, wheat flour, soy flour, pumice, wood flour, walnut shell flour, lignin, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, corn flour, bark flour, wood and nut shell flour, cellulose flour, and mixtures thereof. The agriculturally acceptable carrier can be present in an amount of 99.9% or less, 99% or less, 98% or less, 97% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, by weight, based on the weight of the composition.

[0195] When the composition comprises an amino acid, the amino acid may be provided separately from the amino acids that make up the polypeptide. For example, isolated amino acids may be used. Suitable amino acids include any natural or non-natural amino acids. For example, the composition may comprise cysteine.

[0196] Unless otherwise specified, the content of each agriculturally acceptable adjuvant may be 0.1 to 60 weight %, 0.5 to 50 weight %, or 10 to 30 weight % of the total weight of the composition.

[0197] When the composition includes a preservative, the preservative may include preservatives based on dichlorophenol and benzyl alcohol hemiformal (PROXEL from ICI or ACTICIDE RS from Thor Chemie and KATHON MK from Dow Chemical) and isothiazolinone derivatives such as alkylisothiazolinone and benzisothiazolinone (ACTICIDE MBS from Thor Chemie). As further examples, suitable preservatives include MIT (2-methyl-4-isothiazolin-3-one), BIT (1,2-benzisothiazolin-3-one, available from Avecia, Inc. as PROXEL GXL in a sodium hydroxide and dipropylene glycol solution), 5-chloro-2-(4-chlorobenzyl)-3-one, and 1,2-diisothiazolin-3-one. (2H)-isothiazolone, 5-chloro-2-methyl-2H-isothiazol-3-one, 5-chloro-2-methyl-2H-isothiazol-3-one, 5-chloro-2-methyl-2H-isothiazol-3-one hydrochloride, 4,5-dichloro-2-cyclohexyl-4-isothiazolin-3-one, 4,5-dichloro-2-octyl-2H-isothiazol-3-one, 2-methyl-2H-isothiazol-3-one, 2-methyl-2H-isothiazol-3-one-calcium chloride complex, 2-octyl-2H-isothiazol-3-one, benzyl alcohol hemiformal, or any combination thereof.

[0198] When the composition comprises a buffer, the buffer may include potassium, phosphate, phosphate, citric acid, citrate, sulfate, MOPS or HEPES. The buffer may stabilize the polypeptide in the composition.

[0199] When the composition includes a wetting agent, the wetting agent may include silicones, polyoxyethoxylates, polysorbates, polyethylene glycols and their derivatives, ethoxylates, crop oils, and polysaccharides.

[0200] When the composition includes a surfactant, the surfactant may include heavy petroleum, heavy petroleum distillates, polyol fatty acid esters, polyethoxylated fatty acid esters, aryl alkyl polyoxyethylene glycols, polyoxyethylene polyoxypropylene monobutyl ethers, alkylamine acetates, alkylaryl sulfonates, polyols, alkyl phosphates, alcohol ethoxylates, alkylphenol ethoxylates, alkylphenol ethoxylates, alkoxylated polyols, alkyl polyethoxy ethers, alkyl polyoxyethylene glycerols, ethoxylates and soybean oil derivatives, silicone-based surfactants, or any combination thereof. The surfactant may be included in a range of compositions including foliar application compositions.

[0201] When the composition comprises a coating agent, the coating agent may include an adhesion promoter, a polymer, a filler, or an extender.

[0202] Viscosity enhancers may include, but are not limited to, carboxymethylcellulose, and natural and synthetic polymers in powder, granule or latex form, such as gum arabic, chitin, polyvinyl alcohol and polyvinyl acetate, and natural phospholipids, such as cephalin and lecithin, and synthetic phospholipids. Viscosity enhancers include those preferably composed of adhesive polymers, which may be natural or synthetic and have no phytotoxic effect on the seeds to be coated. Additional viscosity increasing agents that may be included alone or in combination include, for example, polyesters, polyetheresters, polyanhydrides, polyester polyurethanes, polyesteramides; polyvinyl acetate; polyvinyl acetate copolymers; polyvinyl alcohol and tylose; polyvinyl alcohol copolymers; polyvinyl pyrrolidone; polysaccharides, including starch, modified starch and starch derivatives, dextrins, maltodextrins, alginates, chitosan and cellulose, cellulose esters, cellulose ethers and cellulose ether esters, including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose; fats; oils; proteins, including casein, gelatin and zein; gum arabic; shellac; vinylidene chloride and vinylidene chloride copolymers; lignin sulfonates, particularly calcium lignin sulfonate; polyacrylates, polymethacrylates and acrylic acid copolymers; polyvinyl acrylates; polyethylene oxides; polybutenes, polyisobutylenes, polystyrenes, polybutadienes, polyvinylamines, polyvinylamides; acrylamide polymers and copolymers; polyhydroxyethyl acrylates, methacrylamide monomers; and polychloroprene, or any combination thereof. Tackifiers are useful in a variety of compositions, including compositions for seed treatment.

[0203] When the composition includes an abrasive, the abrasive may include talc, graphite, or a combination of both.

[0204] Wetting agent is a hygroscopic substance that helps to keep moisture. When the composition includes wetting agent, the wetting agent can include: glycerol, glycerol, glycerol derivatives (such as monostearate, triacetin, triacetin, propylene glycol, hexylene glycol or butylene glycol), triethylene glycol, tripropylene glycol, triacetin, sucrose, tagatose, sugar alcohol or sugar polyol (such as glycerol, sorbitol, xylitol, mannitol or mannitol), polymerized polyol (such as polydextrose, collagen, aloe or aloe gel) or alpha hydroxy acid (such as lactic acid, honey, molasses, saponaria, sodium hexametaphosphate, lithium chloride or urea). Synthetic wetting agent can also include: butylene glycol and Tremella extract.

[0205] When the compounds described herein are formulated or applied in combination with commercially available fungicides, the compositions can provide an additional layer of protection to enhance the prevention or spread of disease in plants.

[0206] A variety of colorants can be used, including organic chromophores classified as nitroso, nitro, azo (including monoazo, disazo and polyazo), diphenylmethane, triarylmethane, xanthene, methane, acridine, thiazole, thiazine, indoleamine, indophenol, azine, oxazine, anthraquinone, phthalocyanine or any combination thereof.

[0207] Biological control agents are broadly defined as microorganisms that can replace synthetic pesticides or fertilizers. When the composition includes a biological control agent, the biological control agent can include Bacillus thuringiensis, Bacillus megaterium, Bacillus mycoides isolate J, Bacillus methylotrophicus, Bacillus floridus, Chromobacterium subtsugae, Delftia acidovorans, Streptomyces lydicus, Streptomyces columbia, Streptomyces flavosus K61, Penicillium bilais, a lipopeptide-producing Bacillus subtilis strain, a lipopeptide-producing Bacillus amyloliquefaciens strain, a Bacillus firmus strain, or a Bacillus pumilus strain.

[0208] When the composition includes a fertilizer, the fertilizer can include ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcite limestone, calcium oxide, calcium nitrate, dolomite limestone, slaked lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrate, magnesian limestone, magnesium oxide, urea, urea formaldehyde, urea ammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylene diurea, K 2 SO4-Mg 2 SO 4 , potash alum, sylvite, kieserite, Epsom salt, elemental sulfur, marl, ground oyster shells, fish meal, oil cake, fish manure, blood meal, phosphate rock, superphosphate, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, greensand, cottonseed meal, feather meal, crab meal, fish emulsion, humic acid, or any combination thereof.

[0209] The fertilizer may include a liquid fertilizer or a dry fertilizer. The composition may include a micronutrient fertilizer material including boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, sodium tetraborate decahydrate, iron sulfate, iron oxide, ammonium iron sulfate, iron frit, iron chelate, manganese sulfate, manganese oxide, manganese chelate, manganese chloride, manganese frit, sodium molybdate, molybdic acid, zinc sulfate, zinc oxide, zinc carbonate, zinc frit, zinc phosphate, zinc chelate, or any combination thereof.

[0210] When the composition comprises a biostimulant, the biostimulant may include seaweed extracts, inducers, polysaccharides, monosaccharides, protein extracts, soybean extracts, humic acid, plant hormones, plant growth regulators, or any combination thereof.

[0211] Examples of thickeners, i.e. compounds which impart improved flow properties to the formulation (i.e. high viscosity under static conditions and low viscosity during stirring), are polysaccharides and organic and inorganic clays, such as xanthan gum (Kelzan®, CP Kelco, USA), Rhodopol® 23 (Rhodia, France), Veegum® (RT Vanderbilt, USA) or Attaclay® (Engelhard Corp., NJ, USA).

[0212] Examples of suitable antifreeze agents are ethylene glycol, propylene glycol, urea and glycerol.

[0213] Examples of defoamers are silicone emulsions (e.g., Silikon® SRE, Wacker, Germany and Rhodorsil®, Rhodia, France), long-chain alcohols, fatty acids, fatty acid salts, fluorinated organic compounds, and mixtures thereof.

[0214] Fungicides may include aldimorph, ampropylfos, ampropylfos potassium, andoprim, anilazine, azaconazole, azoxystrobin, benalaxyl, benodanil, benomyl, benzamacril, benzamacril-isobutyl, benzovindflupyr, bialaphos, binapacryl, biphenyl, bitertanol, blasticidin-S, boscalid, bromuconazole, bupirimate, buthiobate, calcium polysulphide, capsimycin, captafol, captan, carbendazim, carvon, quinomethionate, chlorothiazide benthiazone), chlorfenazole, chloroneb, chloropicrin, chlorothalonil, chlozolinate, clozylacon, cufraneb, cymoxanil, cyproconazole, cyprodinil, cyprofuram, debacarb, dichlorophen, dichlorophen diclobutrazole, diclofluanid, diclomezine, dicloran, diethofencarb, dimethirimol, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinocap, diphenylamine, dipyrithione,ditalimfos, dithianon, dodemorph, dodine, drazoxolon, edifenphos, epoxiconazole, etaconazole, ethirimol, etridiazole, famoxadon, fenapanil, fenarimol, fenbuconazole, fenfuram, fenitropan, fenpiclonil, fenpropidin, fenpropimorph, fentin acetate acetate), fentinhydroxide, ferbam, ferimzone, fluazinam, fludioxonil, flumetover, fluoromide, fluoxastrobinfluquinconazole, flurprimidol, flusilazole, flusulfamide, flutolanil, flutriafol, folpet, fosetyl-aluminium, fosetylated sodium l-sodium), fthalide, fuberidazole, furaxyl, furametpyr, furcarbonil, furconazole, furconazole-cis, furmecyclox, guazatine, hexachlorobenzene, hexaconazole, hymexazole, imazalil, imibenconazole, iminoctadine, iminoctadine albesilate, iminoctadine triacetate, iodocarb, IBP, iprodione,Irumamycin, isoprothiolane, isovaledione, kasugamycin, kresoxim-methyl, copper preparations, for example, copper hydroxide, copper cyclohexane, copper oxychloride, copper sulfate, copper oxide, copper quinoline and Bordeaux mixtures, mancopper, mancozeb, maneb, meferimzone, mepanipyrim, mepronil, metconazole, metalzxyl, methasulfocarb, methfuroxam, metiram, metomeclam, metsulfovax, mildiomycin, myclobutanil tanil), myclozolin, nickel dimethyl dithiocarbamate, nitrosyl isopropyl ester, flufenamic acid, furamide, oxadixyl, oxamyl, oxolinic acid, oxycarboxin, oxycontin, paclobutrazol, pyraclostrobin, penconazole, pencuron, chlorpyrifos, picoxystrobin, pimaricin, powdery mildew, polyoxin, polyoxin, allylbenzazole, prochloraz, procymidone, propamocarb, propanosine-sodium, propiconazole, propineb, prothioconazole, pyraclofos, pyrimidine oxime, pyrimethanil, pyrimidine ... Quinone, clopidogrel, quinazoline, pentachloronitrobenzene (PCNB), methoxyacrylate, sulfur and sulfur preparations, tebuconazole, chloranil, tetrachloronitrobenzene, tetracyclazole, tetrafluconazole, thiabendazole, thiabendazole, thiophanate-methyl, thiophanate-methyl, thioxanil, methyl tolclofos, p-toluenesulfonyl fluoride, triadimefon, triadimenol, triazoline, triazole, triazine, salicylate, tricyclazole, triclopyr, trimorph, trifloxystrobin, triflumizole, methomyl, uniconazole, jinggangmycin A, vinclozolin, voriconazole, zaliram, mancozeb, ziram, and Dagger G, OK-8705, OK-8801, a-(1,1-dimethylethyl)-(3-(2-phenoxyethyl)-1H-1,2,4-triazole-1-ethanol, a-(2,4-dichlorophenyl)-[3-fluoro-3-propyl-1H-1,2,4-triazole-1-ethanol, a-(2,4-dichlorophenyl)-[3-methoxy-a-methyl-1H-1,2,4-triazole-1-ethanol, a-(5-methyl-1,3-dioxane-5-yl)-[3-[[4-(trifluoromethyl)-phenyl]-methylene]-1H-1,2,4-triazole-1-ethanol,(5RS,6RS)-6-hydroxy-2,2,7,7-tetramethyl-5-(1H-1,2,4-triazol-1-yl)-3-octanone, (E)-a-(methoxyimino)-N-methyl-2-phenoxy-phenylacetamide, 1-isopropyl{2-methyl-1-[[[1-(4-methylphenyl)-ethyl]-amino]-carbonyl]-propyl}carbamate, 1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-ethanone-O-(phenylmethyl)-oxime, 1-(2-methyl-1-naphthyl)-1H-pyrrole-2,5-dione, 1-(3,5-dichlorophenyl)-3-(2-propenyl)-2,5-pyrrolidinedione, 1-[(diiodomethyl)- sulfonyl]-4-methyl-benzene, 1-[[2-(2,4-dichlorophenyl)-1,3-dioxolan-2-yl]-methyl]-1H-imidazole, 1-[[2-(4-chlorophenyl)-3-phenyloxiranyl]-methyl]-1H-1,2,4-triazole, 1-[1-[2-[(2,4-dichlorophenyl)-methoxy]-phenyl]-vinyl]-1H-imidazole, 1-methyl-5-nonyl-2-(phenylmethyl)-3-pyrrolidinol, 2′,6′-dibromo-2-methyl-4′-trifluoromethoxy-4′-trifluoromethyl-1,3-thiazole-carboxanilide, 2,2-dichloro-N-[1-(4-chlorophenyl)-ethyl]-1-ethyl-3-methyl-cyclopropanecarboxamide, 2,6 -dichloro-5-(methylthio)-4-pyrimidinyl-thiocyanate, 2,6-dichloro-N-(4-trifluoromethylbenzyl)-benzamide, 2,6-dichloro-N-[[4-(trifluoromethyl)-phenyl]-methyl]-benzamide, 2-(2,3,3-triiodo-2-propenyl)-2H-tetrazole, 2-[(1-methylethyl)-sulfonyl]-5-(trichloromethyl)-1,3,4-thiadiazole, 2-[[6-deoxy-4-O-(4-O-methyl-(3-D-pyranosyl)-aD-pyranosyl]-amino]-4-methoxy-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile, 2-aminobutane, 2-bromo-2-(bromomethyl)-glutaronitrile, 2-chloro-N-(2, 3-dihydro-1,1,3-trimethyl-1H-inden-4-yl)-3-pyridinecarboxamide, 2-chloro-N-(2,6-dimethylphenyl)-N-(isothiocyanate methyl)-acetamide, 2-phenylphenol (OPP), 3,4-dichloro-1-[4-(difluoromethoxy)-phenyl]-pyrrole-2,5-dione, 3,5-dichloro-N-[cyano[(1-methyl-2-propargyl)-oxy]-methyl]-benzamide, 3-(1,1-dimethylpropyl-1-oxo-1H-indene-2-carbonitrile, 3-[2-(4-chlorophenyl)-5-ethoxy-3-isoxazolidinyl]-pyridine, 4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)-1H-imidazole-1-sulfonamide,4-Methyltetrazo[1,5-a]quinazolin-5(4H)-one, 8-(1,1-dimethylethyl)-N-ethyl-N-propyl-1,4-dioxaspiro[4, 5] decane-2-methylamine, 8-hydroxyquinoline sulfate, 9H-xanthene-2-[(phenylamino)-carbonyl]-9-carboxylic acid hydrazide, bis-(1-methylethyl)-3-methyl-4-[(3-methylbenzoyl)-oxy]-2,5-thiophene dicarboxylate, cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-cycloheptanol, cis-4-[3-[4-(1,1-dimethylpropyl)-phenyl-2-methylpropyl]-2,6-dimethyl-morpholine hydrochloride, ethyl [(4-chlorophenyl)-azo]-cyanoacetate, potassium bicarbonate, methanetetrathiol sodium salt, 1-(2,3-dihydro-2,2-dimethyl N-(2,6-dimethylphenyl)-N-(5-isoxazolylcarbonyl)-DL-alanine methyl ester, N-(chloroacetyl)-N-(2,6-dimethylphenyl)-DL-alanine methyl ester, N-(2,3-dichloro-4-hydroxyphenyl)-1-methyl-cyclohexanecarboxamide, N-(2,6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-furanyl)-acetamide, N-(2,6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-thienyl)-acetamide, N-(2-chloro-4-nitrophenyl)-4-methyl-3 -nitrobenzenesulfonamide, N-(4-cyclohexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine, N-(4-hexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine, N-(5-chloro-2-methylphenyl)-2-methoxy-N-(2-oxo-3-oxazolidinyl)-acetamide, N-(6-methoxy)-3-pyridyl)-cyclopropanecarboxamide, N-[2,2,2-trichloro-1-[(chloroacetyl)-amino]-ethyl]-benzamide, N-[3-chloro-4,5-bis(2-propynyloxy)-phenyl]-N′-methoxy-formamidineamide, N-formyl-N-hydroxy-DL-alanine sodium salt, O,O -diethyl[2-(dipropylamino)-2-oxoethyl]-ethylphosphorothioamide, O-methyl S-phenylphenylpropylphosphorothioamide, S-methyl 1,2,3-benzothiadiazole-7-thioester, spiro[2H]-1-benzopyran-2,1′(3′H)-isobenzofuran]-3′-one, N-trichloromethyl)thio-4-cyclohexane-1,2-dicarboximide, tetramethylthioperoxydicarbonic acid diamide, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine methyl ester, 4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1-H-pyrrole-3-carbonitrile, or any combination thereof.

[0215] The strobilurin fungicide may include strobilurin A, strobilurin B, strobilurin C, strobilurin D, strobilurin E, strobilurin F, strobilurin G, strobilurin H, azoxystrobin, trifloxystrobin, kresoxim-methyl, fluoxastrobin, picoxystrobin, or any combination thereof.

[0216] Strobilurin fungicides can include non-naturally occurring strobilurin fungicides such as azoxystrobin, trifloxystrobin, kresoxim-methyl, fluoxastrobin or any combination thereof. For example, the strobilurin fungicides can include trifloxystrobin, fluoxastrobin or picoxystrobin. Strobilurin fungicides are used to control a variety of fungal diseases, including saprolegniasis, downy mildew, powdery mildew, leaf spot and wilt fungi, fruit rot and rust. They can be used to treat a variety of crops, including cereals, field crops, fruits, nuts, vegetables, turfgrasses and ornamental plants.

[0217] The triazole fungicide may include prothioconazole, imidazole, imidazil, prochloraz, propiconazole, trifloxystrobin, diniconazole, flusilazole, penconazole, hexaconazole, cyproconazole, myclobutanil, tebuconazole, difenoconazole, tetrafluconazole, fenbuconazole, epoxiconazole, metconazole, fluquinconazole, pentoconazole or any combination thereof.

[0218] In addition, the fungicide may include azoxystrobin, carboxin, difenoconazole, fludioxonil, fluopicolide, ipconazole, metalaxyl-M, pyraclostrobin, silthiopyrad, fluazifop-butyl, thiram, pentothiopyrad, or any combination thereof.

[0219] The herbicides may include 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulfuronmethyl bensulide, bentazon, bispyribac sodium, bromacil, bromoxynil, butylate, carfentrazone, chlorimuron, 2-chlorophenoxy acetic acid, chlorsulfuron, chlorimuron ethyl, ethyl), clethodim, clomazone, clopyralid, cloransulam, CMPP-P-DMA, cycloate, DCPA, desmedipham, dicamba, dichlobenil, diclofop, 2,4-dichlorophenol, dichlorophenoxyacetic acid, dichlorprop, dichlorprop-P, diclosulam, diflufenzopyr, dimethenamid, Dimethylamine salt of 2,4-dichlorophenoxyacetic acid, diquat, diuron, DSMA, endothall, EPTC, ethalfluralin, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fluorxypyr 1-methylheptyl1-methyleptylester), fomesafen, fomesafen sodium salt, foramsulfuron, glufosinate, glufosinate-ammonium, glyphosate, halosulfuron, halosulfuron-methyl, hexazinone, 2-hydroxyphenoxyacetic acid, 4-hydroxyphenoxyacetic acid, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaben, isoxaflutole, lactofen, linuron, mazapyr, MCPA, MCPB, methyl mecoprop, mecoprop-P, mesotrione, metolachlor-S, metribuzin, metsulfuron, metsulfuron-methyl, molinate, MSMA, naproxenamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, nonanoic acid, pendimethalin, phenmedipham, picloram, primsulfuronisulfuron), prodiamine, prometryn, pronamide, propanil, prosulfuron, pyrazon, pyrithiobac, pyroxasulfone, quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulsulfuron fosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thifensulfuron-methyl, thiobencarb, tralkoxydim, triallate, triasulfuron, tribenuron, tribernuron-methyl, triclopyr, trifluralin, triflusulfuron, or any combination thereof.

[0220] When the composition comprises a nematicide, the nematicide may include Bacillus firmus, fluopyram, antibiotic nematicides such as abamectin; carbamate nematicides such as acetoprole, Bacillus chitonosporus, chloropicrin, benclothiaz, benomyl, Burholderia cepacia, cepacia), carbofuran, carbosulfan, and cleothocard; dazomet, DBCP, DCIP, alanycarb, aldicarb, aldoxycarb, oxamyl, diamidafos, fenamiphos, fosthietan, phosphamidon, cadusafos, Chlorpyrifos, diclofenthion, dimethoate, ethoprophos, fensulfothion, fostiazate, harpins, heterophos, imicyafos, isamidofos, isazofos, methomyl, mecarphon, Myrothecium verrucaria), Paecilomyces lilacinus, Pasteurella cedarii (including spores thereof), phorate, phosphocarb, terbufos, thionazin, triazophos, tioxazafen, dazomet, 1,2-dichloropropane, 1,3-dichloropropylene, furfural, iodomethane, metam, methyl bromide, isothiocyanate, xylenols, or any combination thereof. For example, the nematicide may include Bacillus firmus strain i-2580, Pasteurella cedarii (including spores thereof), or fluopyram.

[0221] When the composition comprises a bactericide, the bactericide may include streptomycin, penicillin, tetracycline, oxytetracycline, kasugamycin, ampicillin, oxolinic acid, chlortetracycline, copper oxide, or any combination thereof. For example, the bactericide may include oxytetracycline.

[0222] When the composition comprises an insecticide, the insecticide can include clothianidin, imidacloprid, organophosphate, carbamate, pyrethroid, acaricide, alkyl phthalate, boric acid, borate, fluoride, sulfur, urea substituted with halogenated aromatics, hydrocarbon esters, bio-based insecticides or any combination thereof. For example, the insecticide can include clothianidin or imidacloprid.

[0223] When the composition comprises a pesticide, the pesticide can include an organophosphate, a carbamate, a pyrethroid, a miticide, an alkyl phthalate, boric acid, a borate, a fluoride, sulfur, a halogenated aromatic substituted urea, a hydrocarbon ester, a bio-based pesticide, or any combination thereof.

[0224] Also disclosed is a medicine box, which includes a compound disclosed herein in one or more containers. The disclosed medicine box may optionally include an agriculturally acceptable carrier and / or diluent. In one embodiment, the medicine box includes one or more other components, auxiliary agents or adjuvants as described herein. In one embodiment, the medicine box includes instructions or packaging materials describing how to use the compound or composition of the medicine box. The container of the medicine box can be made of any suitable material, such as glass, plastic, metal, etc., and has any suitable size, shape or structure. In one embodiment, the compound disclosed herein and / or agent are provided in the medicine box as a solid (such as tablet, pill or powder form). In another embodiment, the compound disclosed herein and / or agent are provided in the medicine box as a liquid or solution.

[0225] A number of embodiments of the present invention have been described. It should be understood, however, that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, other embodiments are within the scope of the following claims. Example

[0226] The following examples are presented in order to provide those of ordinary skill in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, articles, devices and / or methods claimed herein, and are intended to be purely exemplary of the present invention and are not intended to limit the scope of what the inventors regard as their invention. However, in light of this disclosure, those skilled in the art will appreciate that many changes can be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the present invention.

[0227] Efforts have been made to ensure accuracy with respect to numbers (eg, amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.

[0228] Peptides and proteins are usually impermeable to plant cell membranes. MTD4 is an engineered human membrane translocation domain that can efficiently enter the cytoplasm of plant cells. Fusion of the MTD4 gene to HrpZ, a bacterial harpin, resulted in efficient delivery of HrpZ into tobacco and tomato plants, causing cell death and enhancing resistance to pathogens.

[0229] To explore whether MTD4 can penetrate into plant tissues and cells, MTD4 and the parent protein (FN3) were expressed and purified from Escherichia coli and labeled with tetramethylrhodamine (TMR) (Liss, V., et al., Sci Rep 5(2016)). TMR and FN3 TMR The MTD4 was sprayed onto leaves of Nicotiana benthamiana that had been previously infiltrated with Agrobacterium expressing green fluorescent protein (GFP). Two hours after spraying, confocal microscopy imaging showed that the MTD4 TMR There is a strong red fluorescence in the cells of the treated Nicotiana benthamiana leaves ( Figure 3B , right), but with FN3 TMR There is no strong red fluorescence in the treated Nicotiana benthamiana leaf cells ( Figure 3B , left). As expected, GFP was expressed and localized in the cytoplasm of cells. TMR Co-localization of green and red fluorescence signals at the cell membrane and in the cytoplasm of sprayed leaves ( Figure 3B , lower right panel) demonstrated that MTD4 effectively entered the cytoplasm of Nicotiana benthamiana cells.

[0230] Harpin proteins are produced by Gram-negative bacteria and secreted through the type III secretion system, acting as inducers of hypersensitive reactions (cell death), immune responses, and plant growth promoters (Choi, M. et al., Molecular Plant-Microbe Interactions 26, 1115-1122 (2013)). These unique properties make harpin proteins ideal defense activators and growth promoters for crops. In fact, over the past two decades, harpin-related products have been developed for use in crop plants. However, despite their effectiveness in certain applications, these products have not yet been widely used in crop production because the proteins cannot effectively penetrate plant tissues and therefore have very low bioavailability when applied on the leaves (Nadendla, SR et al., Carbohyd Polym 199, 11-19 (2018)).

[0231] To test whether MTD4 can improve the permeability of cargo proteins into plant tissues, the HrpZ (He, SY et al., Cell 73, 1255-1266 (1993)) gene was fused to the C-terminus of MTD4, and the MTD4-HrpZ fusion protein was purified from Escherichia coli. Treatment of tobacco leaves (via foliar application) with 10 μM MTD4-HrpZ (but not HrpZ or MTD4) for 24 hours resulted in massive cell death in the leaves ( Figure 4A , left). In contrast, when tobacco leaves were infiltrated with MTD4-HrpZ, HrpZ, or MTD4, a large amount of cell death was observed in leaves treated with MTD4-HrpZ or HrpZ ( Figure 4A , right), confirming that HrpZ alone has the ability to induce cell death when it enters the cytoplasmic space. It was also found that spraying Arabidopsis plants with MTD4-HrpZ caused a large number of cell deaths on the leaves 24 hours after treatment ( Figure 5 ). These results demonstrate that MTD-HrpZ, but not HrpZ, can readily enter tobacco and Arabidopsis cells and cause cell death upon foliar application.

[0232] Effects of MTD4-HrpZ on plant immunity. MTD4-HrpZ was sprayed on tobacco leaves and stained with 3, 3'-diaminobenzidine (DAB) to detect the levels of reactive oxygen species (ROS) in plant tissues. Treatment with MTD4-HrpZ resulted in significantly more ROS accumulation than treatment with HrpZ ( Figure 4B ). In addition, qRT-PCR analysis showed that the expression of two pathogenicity-related genes NtHSR203 and NtCHN50 in tobacco was significantly upregulated 3 hours after spraying MTD4-HrpZ compared with the genes sprayed with HrpZ ( Figure 4C To test whether MTD4-HrpZ could enhance tobacco resistance to the pathogenic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC-, tobacco plants were sprayed with MTD4-HrpZ, HrpZ, or MTD4, and bacterial titers were measured three days post-inoculation (dpi). Leaves sprayed with MTD4-HrpZ contained approximately one order of magnitude less Pst bacteria than leaves treated with HrpZ or MTD4 ( Figure 4D ).

[0233] Botrytis cinerea is one of the most destructive fungal pathogens that affects many plant species, including tomatoes (Cheung, N., et al., Pathogens 9, 923 (2020)). We tested whether MTD4-HrpZ could protect tomato fruits from attack by Botrytis cinerea. Fresh tomatoes were sprayed with MTD4-HrpZ, HrpZ, or MTD4. After 24 hours, mycelial clumps of Botrytis cinerea were inoculated into the injured parts of the tomatoes. Three days after inoculation, tomatoes treated with MTD4-HrpZ had significantly less growth of Botrytis cinerea than tomatoes treated with HrpZ or MTD4 ( Figure 4E The lesion size of the tomatoes treated with MTD4-HrpZ was half that of the lesions of the two control tomatoes ( Figure 4F ).

[0234] Cell-penetrating peptide / protein (CPP) technology has been shown to significantly improve the efficiency of defense activators entering plant cells. Lysine-containing CPPs have higher cell penetration efficiency in plants than in animal cells, which may be due to differences in lipid composition and membrane surface charge between the two cell types. MTD4 enters plant cells efficiently and significantly improves the efficiency of HrpZ entry into plant cells. Because MTD4 can be genetically fused to any cargo peptide / protein and the resulting fusion protein can be produced on a large scale and cost-effectively by fermentation, it has great potential in delivering defense activators and biostimulants for foliar application and seed treatment in crop production.

[0235] method

[0236] Primers and strains. The primers used in this study are listed in Table 3. The strains and plasmids used in this study are listed in Table 4. All gene constructs were confirmed by sequencing.

[0237] Table 3. List of primer sequences used in the study.

[0238]

[0239] Table 4. List of strains and plasmids used in the study.

[0240]

[0241] Plant material and growth conditions. Tobacco and Arabidopsis seeds were sown in soil pots and plants were grown in a growth chamber at 25°C with an 8 hr / 16 hr photoperiod.

[0242] Design, expression and purification of MTD. The human fibronectin type III (FN3) domain was selected as a scaffold for the membrane translocation domain (MTD) (Koide, A., et al. (1998) The fibronectin type III domain as ascaffold for binding proteins. J. Mol. Biol. 284(4):1141-1151). FN3 is a small (90-100 aa), highly stable protein that has been widely used to develop monomers that bind target proteins with high affinity and specificity (Chandler, PG, et al. (2020) Development and Differentiation in MonobodiesBased on the Fibronectin Type 3 Domain. Cells 9(3):610). Previous studies have demonstrated that several loop regions of FN3 are tolerant to mutations (Steven, A., et al. (2012) Design of novel FN3 domainswith high stability by a consensus sequence approach, Protein Engineering, Design and Selection, 25(3):107–117). In addition, FN3 does not contain any cysteine ​​or disulfide bonds and is therefore stable in the intracellular environment. FN3 can be easily folded into its native form without any physical or chemical assistance and can be produced in high yields in E. coli. Finally, FN3 is derived from an abundant human extracellular protein and is unlikely to trigger any immune response.

[0243] The BC, DE, and FG loops of FN3 have previously been shown to be highly tolerant to sequence mutations. The GDSPAS (SEQ ID NO: 106) sequence of the FG loop was replaced by RRRWWW (SEQ ID NO: 104), resulting in MTD1 (Table 5). Together with the existing arginine residues in the FG loop, this generates a putative CPP motif (R 4 W 3) without changing the loop size. Similarly, the tetrapeptide AVTV of the BC loop was replaced with WWWRRR (SEQ ID NO: 105) to utilize the existing arginine in the loop to form a putative CPP, W 3 R 4 (Table 5). The size of the BC loop in the resulting mutant MTD2 was increased by 2 residues. In order to explore the possibility of transplanting the CPP motif to the other end of FN3, the CPP motif R 4 W 3 MTD3 was obtained by replacing the tripeptide NSP in the CD loop. To test the feasibility of transplanting CPP sequences to two different loops, the relatively hydrophobic tripeptide in the BC loop (VTV) was replaced with WYW, and the hydrophilic motif (GDSPAS; SEQ ID NO: 106) in the FG loop was replaced with RRR to generate MTD4. Finally, MTD5 was generated by switching the WYW and RRR motifs of MTD4. Two mutants, MTD4a and MTD4b, were also generated, which contained only half of the CPP motif in their BC and FG loops, respectively, to test the relative importance of the RRRR and WYW motifs. The WYW motif is more hydrophilic than the WWW motif and has been previously reported as an "endosome escape motif" for cell-permeable antibodies (Kim, J.-S., et al. (2016) Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediate endosomalescape. J. Control. Rel. 235:165–175). The loop insertion mutants were analyzed by the online program Phyre2, and their folded structures were predicted based on sequence homology. All mutants maintained an overall fold similar to wild-type FN3, with the CPP motif displayed on their surface and confined in a "loop-like" topology ( Figure 1 ).

[0244] To further improve the properties of MTD4 (e.g., cell entry efficiency, metabolic stability, and expression yield), the BC and FG loops of FN3 were replaced with different combinations of Y, W, A, and R residues to generate MTD6-10 (Table 5). The overall cell entry efficiency of MTD6-10 was assessed by labeling MTD at a unique C-terminal cysteine ​​using tetramethylrhodamine-5-maleimide (TMR). HeLa cells were treated with TMR-labeled proteins (5 μM) for 2 h and then analyzed by live cell confocal microscopy. MTD7 TMR and MTD9 TMR Shown to be related to MTD4 TMR Similar uptake, while MTD6 TMR 、MTD8TMR and MTD10 TMR showed less cell entry. In addition, the isolation yields of MTD6–10 varied between 0.6 and 6.2 mg / L E. coli cell culture (Table 4). Note that MTD4 and MTD6 differ only slightly in the BC loop sequence (“WYW” vs. “YWW”), but have significant differences in isolation yield (9.4 mg / L vs. 0.9 mg / L) as well as cell entry efficiency. Similarly, exchanging the CPP motif between the BC and FG loops of MTD4 resulted in a poorly expressed and less active variant (MTD5 in Table 5). These results indicate that the correct folding / stability and high cell entry efficiency of MTD require not only the presence of amphipathic CPP motifs, but also their correct presentation on the protein surface.

[0245] The DNA sequence encoding WT FN3 was chemically synthesized and ligated into the prokaryotic expression vector pET-15b. To facilitate protein purification and genetic fusion with cargo proteins, a hexahistidine tag and a thrombin cleavage site were added to the N-terminus of FN3, while a flexible linker sequence (GGSGGSGGS; SEQ ID NO: 107) was added to its C-terminus, followed by a recognition site for the restriction endonuclease SacI and cysteine ​​(Table 6). All loop insertion mutants were generated by a one-step polymerase chain reaction (PCR) method (Qi, D., et al. (2008) A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis. J. Virol. Meth. 149: 85–9020) and expressed in Escherichia coli. Among the mutant proteins, MTD1 failed to produce a large amount of soluble protein, while WT FN3 and MTD2-5 produced soluble proteins in good yields (Table 5). Figure 2A-2B Expression and purification of MTD4 is shown as an example. All proteins were purified to near homogeneity by metal affinity chromatography on a Ni-NTA column.

[0246] Table 5. Loop sequences and expression yields of MTD

[0247]

[0248] *During mutagenesis, underlined residues were deleted and bold residues were inserted.

[0249] Cloning, expression and purification of MTD. All loop insertion mutants were generated by a one-step polymerase chain reaction (PCR) method (Qi, D., id.). The peptide sequence of each construct was confirmed by sequencing the entire coding region of the plasmid DNA (Table 6). Pilot-scale protein expression was performed to check the expression level of the mutant proteins, and all mutants were expressed in 5 mL of Escherichia coli BL21 (DE3) bacterial culture. Induction was performed at 37°C in the presence of 0.25 mM IPTG. The expression level was checked by comparing the total cell lysates before and after induction on SDS gel ( Figure 2A-2B ).

[0250] Table 6. Amino acid sequences of WT FN3, MTD, and MTD-cargo fusions

[0251]

[0252]

[0253] Large-scale expression conditions were the same as those for small-scale expression, and E. coli cells were centrifuged and stored at -80°C. The cells were lysed using lysis buffer (50 mL wash buffer, 0.2 mg / mL lysozyme, 2 mM β-mercaptoethanol, 2 mM PMSF, 2 tablets of Roche complete protease inhibitor cocktail). After the cell palates were evenly resuspended in lysis buffer, the cells were sonicated twice (Amp. 70%). The crude cell lysate was centrifuged (12000 g for 20 minutes) and the soluble cell lysate was collected. Protein purification was performed by using fast protein liquid chromatography (FPLC), and the soluble cell lysate was loaded onto a nickel-NTA column (with 15 mM imidazole). The column was washed thoroughly with wash buffer (50 mM Tris (pH 7.4), 300 mM NaCl, 5% glycerol and 50 mM imidazole). The protein was eluted in 30 minutes with a wash buffer containing a linear gradient of 50–500 mM imidazole (pH 7.4).

[0254] MTD4 plasmid construction. The DNA sequence encoding the tenth FN3 domain and HrpZ was chemically synthesized and ligated into the prokaryotic expression vector pET-15b (MilliporeSigma), which had been linearized using restriction endonucleases NdeI and XhoI. The cloning procedure also resulted in the addition of a flexible linker sequence, (GGS) at the C-terminus of FN3. 3, restriction sites (SacI) and cysteine ​​residues. Site-directed mutagenesis of FN3 was previously described to generate plasmid pET-15b-MTD4 (Qi, D.; Scholthof, K.-BG, A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis. J. Virol. Methods 2008, 149(1), 85-90). The HrpZ gene was amplified by PCR using plasmid DNA as a template and primers containing SacI and XhoI restriction sites at the 5' and 3' ends of the HrpZ sequence, respectively (Table 3). The PCR product was digested with SacI and XhoI restriction enzymes and ligated into plasmid pET-15b-MTD4 linearized with the same two enzymes. All constructs contained a six-histidine tag at the N-terminus to facilitate purification. The authenticity of the DNA constructs was confirmed by restriction mapping and sequencing of the entire coding sequence.

[0255] HrpZ protein sequence SEQ ID NO: 146

[0256] MQSLSLNSSSLQTPAMALVLVRPEAETTGSTSSKALQEVVVKLAEELMRNGQLDDSSPLGKLLAKSMAADGKAGGGIEDVIAALDKLIHEKLGDNFGASADSASGTGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDMPMLNKIAQFMDDNPAQFPKPDSGSWVNE LKEDNFLDGDETAAFRSALDIIGQQLGNQQSDAGSLAGTGGGLGTPSSFSNNSSVMGDPLIDANTGPGDSGNTRGEAGQLIGELIDRGLQSVLAGGGLGTPVNTPQTGTSANGGQSAQDLDQLLGGLLLKGLEATLKDAGQTGTDVQSSAAQIATLLVSTLLQGTRNQAAA

[0257] Based on the HrpZ DNA sequence of GenBank accession number: HM358042.1 (SEQ ID NO: 155)

[0258]

[0259] Expression and purification of recombinant proteins. E. coli BL21(DE3) cells transformed with the appropriate plasmids were grown at 37°C in Luria-Bertani (LB) medium supplemented with 75 µg / mL ampicillin. 600 When the p value reached 0.6, the cells were induced at 37°C for 6 h by adding 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). The cells were pelleted by centrifugation (4000 g, 30 min) and resuspended in lysis buffer (50 mL wash buffer, 0.2 mg / mL lysozyme, 2 mM β-mercaptoethanol, 2 mM PMSF, 2 tablets of Roche complete protease inhibitor cocktail per 1 L of cell culture). The cells were briefly sonicated and the crude cell lysate was centrifuged (12000 g, 20 min). The supernatant was loaded onto a 5 mL HisTrap FF nickel affinity column (Cytiva). The column was washed extensively with wash buffer (20 mM Tris, 300 mM NaCl, and 5% glycerol, adjusted to pH 7.4) and wash buffer plus 50 mM imidazole. The protein was eluted with a linear gradient of 50–500 mM imidazole in wash buffer. The purity of the eluted fractions was assessed by SDS-PAGE, and the pure protein fractions were pooled, concentrated in a centrifugal filter device (Millipore), and dialyzed against wash buffer. The protein concentration was determined by Bradford analysis. The purified protein was supplemented with 30% glycerol, aliquoted, snap frozen in an isopropanol bath and stored at -80 °C.

[0260] Fluorescent labeling of proteins. FN3 and MTD4 stock solutions were passed through desalting spin columns (Bio-rad) to remove β-mercaptoethanol. The proteins were treated with 1 mM tris(2-carboxyethyl)phosphine (TCEP) to ensure that the C-terminal cysteine ​​was in the reduced form. Tetramethylrhodamine-5-maleimide (TMR; 8 equiv.) was dissolved in N,N-dimethylformamide (DMF) and slowly added to the protein solution, and the reaction was allowed to proceed for 2 h at room temperature. The reaction mixture was passed through a desalting spin column to remove excess dye. The fluorescence intensity of the proteins was compared with that of the proteins at 280 nm (λ max ) and 544 nm (λ of TMR max ) to estimate the stoichiometry of dye labeling.

[0261] Subcellular localization assay. The pYBA1132 plasmid containing a green fluorescent protein (GFP) tag was transformed into Agrobacterium strain GV3101 (Yan, X. et al. Mol. Plant Breed 10, 371-379 (2012)) and then used to infiltrate Nicotiana benthamiana leaves. Two days after infiltration, MTD4-Rh or FN3-Rh (10 µM) was sprayed on Nicotiana benthamiana leaves, and the treated leaves were incubated in the dark at 23°C. Two hours after spraying, H 2 O 2 The protein solution on the surface of Nicotiana benthamiana leaves was washed three times. Red and green fluorescence were observed using a confocal laser scanning microscope under excitation at 488 nm and 514 nm, respectively.

[0262] DAB staining. Tobacco leaves were immersed in 3,3'-diaminobenzidine (DAB) staining buffer (10 mM MES, pH 6.5, and 1 mg / mL DAB) at 23°C for 18 h. The leaves were then transferred to 90% (v / v) ethanol at 65°C until transparent.

[0263] qRT-PCR. Total RNA was isolated from tobacco leaves using TRIzol reagent according to the manufacturer's instructions. qRT-PCR was performed to quantify the expression levels of pathogenicity-related genes. Gene transcription levels were normalized to the expression of the reference gene NtActin. Primers used to detect the expression of NtHSR203, NtCHN50, and NtActin were as described previously (Zhang, C., et al. J Exp Bot 70, 5407-5421 (2019)). The experiment was performed in three biological replicates.

[0264] Bacterial inoculation. Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC strain lacking the type III secretion system was used for bacterial inoculation assays with slight modifications as described previously (Zhang, X., et al., Plant Mol Biol 90, 19-31 (2016)). Bacteria were cultured in King's medium containing 25 mg / L rifampicin at 28°C. Fresh overnight bacterial cultures were collected and centrifuged at 4000 rpm for 5 minutes and washed with 10 mM MgCl 2 The suspension was washed twice. The OD600 was measured spectrophotometrically and then washed with 10 mM MgCl 2 The samples were further diluted to 0.2 and infiltrated into four-week-old tobacco leaves. Samples were collected at 0 and 3 dpi. The samples were incubated in 10 mM MgCl 2 The samples were ground in PBS and then serially diluted in King's medium containing 25 mg / L rifampicin. The number of colonies formed was counted two days after spreading on plates.

[0265] Fungal inoculation. Tomatoes were sterilized with 75% (v / v) ethanol and then sprayed with 10 µM MTD4-HrpZ, HrpZ, or MTD4. A small incision was made in the tomato fruit with a sharp blade before inoculation with B. cinerea. B. cinerea was cultured in potato dextrose agar (PDA) medium at 28 °C in an incubator. 24 h after spraying with the protein solution, a plug of B. cinerea hyphae was placed on the wounded site. Disease symptoms were evaluated at 3 dpi.

[0266] Statistical analysis. Multiple comparisons were performed using GraphPad Prism version 8.00 for Windows with one-way ANOVA followed by Dunnett’s multiple comparison test.

[0267] Example 2: MTD4-HrpZ for disease control in crop plants

[0268] To test the application of MTD4-HrpZ for disease control in crop plants, tomato plants were used because it is one of the most popular vegetables in the United States. The optimal concentration of MTD4-HrpZ on tomato plants was first determined, which induced an adequate defense response against the bacterial spot pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 but did not cause cell death. Tomato seedlings (cultivar OH88119) were grown in a growth chamber with a day temperature of 26 °C, a night temperature of 22 °C, and a 12-h light / dark cycle. Four-week-old plants were sprayed with different concentrations of MTD4-HrpZ, HrpZ, and MTD4 and inoculated with Pst 24 h later. Disease symptoms were observed one week after inoculation. Spraying tomato plants with 1 µM MTD4-HrpZ effectively inhibited Pst infestation, whereas MTD4 or HrpZ did not ( Figures 6A-6C ).

[0269] In addition, the effectiveness of MTD4-HrpZ was tested against Xanthomonas euvesicatoria pv. perforans (Xep), the pathogen that causes bacterial spot disease. Similar to the results with Pst, application of 1 µM MTD4-HrpZ provided strong protection against Xep infection in 4-week-old tomato plants. In addition, crude bacterial cell lysate from Escherichia coli cells overproducing MTD4-HrpZ also effectively protected 4-week-old tomato plants against bacterial pathogens. A 3-fold dilution of the crude cell lysate protected tomato plants from Xep infection ( Figures 7A-7D ).

[0270] To determine whether MTD4-HrpZ promotes plant growth, tomato seedlings were sprayed with 0.5 µM MTD4-HrpZ twice (6 and 16 days after germination). Treatment with MTD4-HrpZ increased plant height and fresh weight by 15-20% ( Figure 8 and 9A -9D).

[0271] Collectively, the above results clearly demonstrated that MTD4 significantly increased the entry of HrpZ into plant cells during foliar application, leading to enhanced immunity against bacterial pathogens and growth promotion. sequence

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

Claims

1. A peptide comprising: a membrane translocation domain having one or more cell penetrating peptide motifs, and a cargo moiety linked to the membrane translocation domain, wherein the cargo moiety comprises a plant bioactive moiety, wherein at least one cell penetrating peptide motif is 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues; or wherein at least one cell penetrating peptide motif is 3 to 10 amino acid residues in length and has at least two arginine and / or lysine residues, and at least one other cell penetrating peptide motif is 2 to 8 amino acid residues in length and has at least two hydrophobic residues.

2. The peptide of claim 1, wherein the membrane translocation domain is human fibronectin type III.

3. The peptide of claim 2, wherein the human fibronectin type III has 90% sequence similarity to SEQ ID NO:

118.

4. The peptide of any one of claims 1 to 3, wherein the cell penetrating motif has 3 to 10 consecutive arginine residues.

5. The peptide of any one of claims 1 to 4, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD and FG loops, and one or more of the BC, DE, CD or FG loops have a cell penetrating peptide motif.

6. The peptide of any one of claims 1 to 5, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD and FG loops, and two of the BC, DE, CD or FG loops have a cell penetrating peptide motif.

7. The peptide of any one of claims 1 to 6, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD and FG loops, and the BC and the DE, CD or FG loop have a cell penetrating peptide motif.

8. The peptide of any one of claims 1 to 6, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD and FG loops, and the BC and FG loops have a cell penetrating peptide motif.

9. The peptide of claim 8, wherein the cell penetrating peptide motif in the BC loop has 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, and the cell penetrating peptide motif in the FG loop has 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues.

10. The peptide of any one of claims 1 to 9, wherein the cell penetrating peptide motif in the FG loop has 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, and the cell penetrating peptide motif in the BC loop has 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues.

11. The peptide of any one of claims 1-10, wherein a second cell penetrating peptide motif is present and is WW, FF, WF, FW, WWW, FFF, WFW, FWF, WWF, WFF, FWW, FFW, WYW, WWH, YWW or WYH.

12. The peptide of any one of claims 1-11, wherein the cell penetrating peptide motif is RRRWWW (SEQ ID NO: 104) or WWWRRR (SEQ ID NO: 105).

13. A peptide as described in any one of claims 1 to 12, wherein the peptide comprises TGRRRRWWWSKPI (SEQ ID NO: 111); APWWWRRRRYY (SEQ ID NO: 112); GGRRRRWWWVQE (SEQ ID NO: 113); APAWYWRYY (SEQ ID NO: 114); TGRRRRSKPI (SEQ ID NO: 115); APARRRRYY (SEQ ID NO: 116); TGWYWRSKPI (SEQ ID NO: 117); SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164 or SEQ ID NO:

165.

14. The peptide of any one of claims 1-13, wherein the peptide comprises SEQ ID NO.: 119, 120, 121, 122, 123, 124, 125, 167, 168, 169, 170 or 171.

15. The peptide of any one of claims 1-14, wherein the cargo moiety is linked to the membrane translocation domain at the N-terminus or C-terminus of the membrane translocation domain, or at a side chain within the membrane translocation domain.

16. The peptide of any one of claims 1-15, wherein the cargo moiety further comprises a detectable moiety.

17. A peptide as described in any one of claims 1-16, wherein the peptide comprises SEQ ID NO.:126, 127 or 128.

18. The peptide of any one of claims 1-17, wherein the plant bioactive portion comprises synthetically derived or naturally occurring flagellin and flagellin-related polypeptides (including those conserved in Bacillus), thionine, harpin protein or polypeptide or harpin-like polypeptide, elongation factor Tu (EF-Tu), phytosulfopeptide (PSKα), root hair promoting polypeptide (RHPP), hypersensitive response elicitor protein or polypeptide, or any combination thereof.

19. A composition for delivering a cargo moiety into a plant cell, comprising SEQ ID NO: 122 covalently bound to a cargo moiety, wherein the cargo moiety comprises a plant bioactive moiety.

20. The composition of claim 19, wherein the cargo moiety further comprises a detectable moiety.

21. A composition as described in any one of claims 19 to 20, wherein the plant biologically active part comprises synthetically derived or naturally occurring flagellin and flagellin-related polypeptides, thionines, harpin proteins or polypeptides or harpin-like polypeptides, elongation factor Tu (EF-Tu), phytosulfopeptidase (PSKα), root hair promoting polypeptide (RHPP), hypersensitive response elicitor proteins or polypeptides or any combination thereof.

22. A method of delivering a cargo moiety to a plant, comprising contacting the plant with the peptide of any one of claims 1-18 or the composition of any one of claims 19-21.

23. A method of delivering a plant stimulant to a plant comprising contacting the plant with a peptide as claimed in any one of claims 1 to 18 or a composition as claimed in any one of claims 19 to 21.

24. A method of delivering a plant activator to a plant comprising contacting the plant with the peptide of any one of claims 1-18 or the composition of any one of claims 19-21.

25. A method for protecting plants from biotic stress; stimulating seeds during germination; protecting plants from abiotic stress; enhancing plant growth, yield, health, lifespan, productivity and / or vigor; providing plants with multiple disease resistance; or any combination thereof, the method comprising contacting the plant with a peptide as described in any one of claims 1-18 or a composition as described in any one of claims 19-21.

26. A method for treating a plant suffering from a disease caused by a pathogen, comprising contacting the plant with the peptide of any one of claims 1 to 18 or the composition of any one of claims 19 to 21.

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