Compositions for drug delivery into plant cells - Patents.com

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, the cargo moiety comprising a plant bioactive moiety. At least one cell penetrating peptide motif is 3-10 amino acid residues long and has at least three arginine and / or lysine residues; or at least one cell penetrating peptide motif is 3-10 amino acid residues long and has at least two arginine and / or lysine residues, and at least one other cell penetrating peptide motif is 2-8 amino acid residues long and has at least two hydrophobic residues. Also described are methods of delivering a cargo moiety into a plant cell, comprising contacting the plant cell with a peptide disclosed herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 338,302, filed May 4, 2022, which is incorporated by reference in its entirety.

[0002] Reference to sequence listing This sequence listing, created as text on May 4, 2023 under the name "103361-267WO1_ST26" and submitted the same day, with a file size of 295,920 bytes, is incorporated herein by reference in accordance with 37 C.FR § 1.52(e)(5). [Background technology]

[0003] Fertilizers and pesticides play a vital role in modern agriculture, but their adverse effects on the environment and animal / human health have influenced the rapid growth of organic agriculture in recent years. With a market of approximately $80 billion, it is clear that organic agriculture will become an essential and innovative agricultural system that balances sustainability with food / ecosystem security and human health benefits (Reganold and Wachter, 2016). Promising eco-friendly innovations are the use of (i) biological defense activators that improve plant immunity against pathogens and insects, and (ii) biostimulants that enhance plant growth and resistance to abiotic stresses. Many of the plant defense activators and biostimulants currently used in organic agriculture are proteins / peptides that trigger appropriate signaling pathways, thereby stimulating defense and / or growth. However, a formidable challenge in using these peptides / proteins is their poor penetration efficiency in foliar applications and seed treatments (Nadendla, S.R., 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 De Rossi, D., et al., J Biol Chem 269, 10444-10450 (1994)). Since then, about 2000 CPPs have been reported, the vast majority of which are linear peptides. Despite significant efforts in academia and industry, drug delivery with linear CPPs has been largely unsuccessful, as linear CPPs are proteolytically unstable, have low cytosolic entry efficiency, and exhibit poor pharmacokinetics. Significant progress has come from cyclization of CPPs, as cyclic variants are proteolytically stable and exhibit a 60-fold improved cytosolic entry efficiency compared to their linear counterparts (Qian, Z. et al. Biochemistry 55, 2601-2612 (2016)). Further studies revealed that the potent activity of cyclic CPPs is due to their ability to evade endosomes via a previously unappreciated vesicle budding and collapse pathway (Sahni, A., et al., ACS Chem Biol 15, 2485-2492 (2020)). This understanding then led to the realization that to achieve high cell penetrating activity, CPPs must adopt an appropriate 3D structure. Based on this insight, Bhat et al. recently engineered MTD4, a highly effective CPP derived from the tenth human fibronectin type III (FN3) domain. Unlike cyclic CPPs, which must be chemically synthesized before conjugation to proteins, MTD4 can be genetically fused to any peptide / protein cargo and produced recombinantly.

[0005] Research on CPP-mediated protein delivery in plants was initiated 15 years ago. The first study showed that CPPs were internalized in Nicotiana tabacum protoplasts and demonstrated that these peptides could enter plant cells by transfection (Mae, M., et al. Biochimica et Biophysica Acta (BBA)-Biomembranes 1669, 101-107 (2005)). Later, translocation of various CPPs in wheat immature embryos in the presence of a cell membrane permeabilizing agent was also reported (Chugh, A., et al., FEBS J 275, 2403-2414 (2008)). Successful uptake of protein cargo by live microspore cells was also achieved by utilizing a reversible disulfide bond between the R9 CPP and the mCherry protein (Bilichak, A., et al., Front Plant Sci 6 (2015)). Recently, the penetration efficiency of 55 CPPs (most of which have been previously tested in animals) was evaluated in dicotyledonous and monocotyledonous plants, and several CPPs were found to enter plant cells (Numata, K., et al., SCI REP-UK 8 (2018)). An investigation 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 have been no reports of using CPPs to deliver either defense-promoting 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] Disclosed herein are compounds, compositions, and methods for making and using such compounds and compositions. In one aspect, disclosed 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, the cargo moiety comprising a plant bioactive moiety, wherein at least one cell penetrating peptide motif is 3-10 amino acid residues in length and has at least three arginine and / or lysine residues, or at least one cell penetrating peptide motif is 3-10 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 2-8 amino acid residues in length and has at least two hydrophobic residues. Also disclosed are methods of delivering a plant bioactive moiety into a plant cell, the methods comprising contacting the 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 the PDB file 1ttg, and the structures of MTD1-5 were predicted by Phyre2. The inserted CPP motifs of MTD1-5 are highlighted in black. [Figure 2A] 1 shows expression and purification of MTD4. FPLC chromatogram showing elution of MTD4 from a Ni-NTA column (MTD4 elutes as a broad peak). [Figure 2B]Figure 1: Expression and purification of MTD4. SDS-PAGE showing the expression level and different fractions during purification on 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). [Figure 3A] The MTD4 structure is shown. [Figure 3B] Figure 1 shows MTD4 protein permeability in N. benthamiana leaves. Two days after GFP infiltration, 10 μM rhodamine-labeled MTD4 (MTD4-Rh) or FN3 (FN3-Rh) was sprayed onto N. benthamiana leaves. Two hours after spraying, the sprayed leaf area was washed with H2O2 to remove residual protein, and then red (Rh) and green fluorescence (GFP) were measured using a confocal microscope. [Figure 4A] Figure 1 shows the enhancement of HrpZ permeability and disease resistance by MTD4 in tobacco and tomato. Images showing cell death caused by MTD4-HrpZ treatment. 2 ml of 10 μM MTD4-HrpZ, HrpZ, or MTD4 recombinant protein was placed on the leaves of 6-week-old N. tabacum plants. Images shown were taken 24 hours after the respective protein treatment. [Figure 4B] Figure 1 shows the enhancement of HrpZ permeability and disease resistance by MTD4 in tobacco and tomato. Images of reactive oxygen species (ROS) accumulation after MTD4-HrpZ treatment are shown. MTD4-HrpZ, HrpZ, or MTD4 (10 μM) were sprayed onto 1-month-old N. tabacum leaves. ROS accumulation was detected by 3,3'-diaminobenzidine (DAB) staining. Images were taken 12 hours after treatment with the respective proteins. [Figure 4C] Figure 1 shows the enhancement of HrpZ permeability and disease resistance by MTD4 in tobacco and tomato. Graph showing qRT-PCR analysis of the expression levels of NtHSR203 after treatment with MTD4-HrpZ, HrpZ or MTD4 protein. Letters (a, b, c) indicate significant differences (P<0.05, Dunnett's multiple range test). [Figure 4D] Figure 1 shows the enhancement of HrpZ permeability and disease resistance by MTD4 in tobacco and tomato. Graph showing qRT-PCR analysis of the expression levels of NtCHN50 after treatment with MTD4-HrpZ, HrpZ or MTD4 protein. Letters (a, b, c) indicate significant differences (P<0.05, Dunnett's multiple range test). [Figure 4E] Figure 1 shows the permeability of HrpZ and the enhancement of disease resistance by MTD4 in tobacco and tomato. Figure 2 shows the enhanced resistance in plants treated with MTD4-HrpZ. MTD4-HrpZ, HrpZ, or MTD4 protein (2 μM) was sprayed on N. tabacum leaves. 12 h after spraying, the leaves were pressure inoculated with Pst DC3000 hrcC- at OD600 ~ 0.2. In plants, bacterial numbers were assessed 0 and 3 days after inoculation. Here, they are shown as colony forming units (cfu). Data shown are mean ± SEM, a and b indicate statistically significant differences (Duncan's multiple range test, P < 0.05). [Figure 4F] Figure 1. Enhancement of HrpZ penetration and disease resistance by MTD4 in tobacco and tomato. Images showing disease symptoms in tomato treated with MTD4-HrpZ, HrpZ or MTD4 after inoculation with B. cinerea. MTD4-HrpZ, HrpZ or MTD4 protein (5 μM) was sprayed onto disinfected tobacco surfaces. Blocks of B. cinerea were placed on small wounds on tomato fruit after 24 h of protein treatment. Pictures were taken at 3 dpi. [Figure 4G] Figure 1 shows the permeability of HrpZ and the enhancement of disease resistance by MTD4 in tobacco and tomato.Figure 2 shows a graph showing the diameter of the infection zone measured at 3 dpi. [Diagram 5] 13 is an image showing cell death caused by MTD4-HrpZ in Arabidopsis following application to leaves of 6-week-old plants. Images were taken 24 hours after treatment. [Figure 6A] 13 is an image showing that MTD4-HrpZ confers resistance to the tomato bacterial spot pathogen. Image after application of MTDA4, 1 μM. [Figure 6B]13 is an image showing that MTD4-HrpZ confers resistance to the tomato bacterial spot pathogen. Image after HrpZ application, 1 μM. [Figure 6C] 13 is an image showing that MTD4-HrpZ confers resistance to the tomato bacterial spot pathogen. Image after application of MTD4-HrpZ, 1 μM. [Figure 7] 13A-13D are images showing that MTD4-HrpZ confers resistance to tomato bacterial spot pathogens. A, images after application of MTD4. B, images after application of MTD4-HrpZ at different cell lysate dilutions. 3-fold dilution of cell lysate. C, images after application of MTD4-HrpZ at different cell lysate dilutions. 4-fold dilution of cell lysate. D, images after application of MTD4-HrpZ at different cell lysate dilutions. 5-fold dilution of cell lysate. [Figure 8] Image showing that MTD4-HrpZ promotes tomato growth. Data were obtained from one experiment with two replicates, where plants were sprayed twice with the three proteins. [Figure 9A] Graph showing that MTD4-HrpZ promotes tomato growth. Graph of plant height to shoot tip for MTD4, HrpZ, and MTD4-HrpZ. Letters (a, b, c) indicate significant differences (P<0.05, Dunnett's multiple range test). [Figure 9B] Graph showing that MTD4-HrpZ promotes tomato growth. Graph of plant height to leaf tip for MTD4, HrpZ, and MTD4-HrpZ. Letters (a, b, c) indicate significant differences (P<0.05, Dunnett's multiple range test). [Figure 9C] Graph showing that MTD4-HrpZ promotes tomato growth. Graph of fresh weight (g) of MTD4, HrpZ, and MTD4-HrpZ. Letters (a, b, c) indicate significant differences (P<0.05, Dunnett's multiple range test). [Figure 9D]Graph showing that MTD4-HrpZ promotes tomato growth. Graph of dry weight (g) of MTD4, HrpZ, and MTD4-HrpZ. Letters (a, b, c) indicate significant differences (P<0.05, Dunnett's multiple range test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention may be understood more readily by reference to the following detailed description of the invention and the examples contained herein.

[0012] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it should be understood that they are not limited to specific synthetic methods, unless otherwise specified, or to specific reagents, unless otherwise specified, and therefore can, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described.

[0013] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided herein may be different from the actual publication dates, which may require independent confirmation.

[0014] definition Throughout this specification, the terms "about" and / or "approximately" may be used in conjunction with numerical values ​​and / or ranges. The term "about" is understood to mean a value close to the recited value, as well as the recited value.

[0015] Throughout this specification, numerical ranges are provided for specific quantities. These ranges should be understood to include all values ​​and subranges therein. Thus, the range "50-80" 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.). Furthermore, all values ​​within a given range may be endpoints of the range encompassed thereby (e.g., the range 50-80 includes ranges having endpoints such as 55-80, 50-75, etc.).

[0016] The term "a" or "an" refers to one or more of that entity, for example, a "polypeptide conjugate" refers to one or more polypeptide conjugates or at least one polypeptide conjugate. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. Furthermore, reference to a "polypeptide conjugate" by the indefinite article "a" or "an" does not exclude the possibility that there is a plurality of polypeptide conjugates, unless the context clearly requires that there is one and only one.

[0017] As used herein, the term "adjacent" refers to two adjacent amino acids that are connected by a covalent bond. "Adjacent" is also used synonymously with "consecutive."

[0018] The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize the degeneracy of the active ingredient and to minimize side effects in the subject.

[0019] As used herein, a "cell-penetrating peptide" or "CPP" refers to any peptide, including a protein (i.e., a polypeptide), that can permeate a cell membrane. As used herein, a "cyclic cell-penetrating peptide" or "cCPP" refers to any cyclic peptide that can permeate a cell membrane.

[0020] As used herein, "foliar treatment" refers to a composition that is applied to the above-ground or foliage parts of a plant or plant part, which may include leaves, stems, flowers, branches, or any aerial plant parts, e.g., tips.

[0021] 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 may 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 unnatural amino acid or polypeptide. In other embodiments, the linker is a synthetic compound that contains two or more suitable functional groups suitable for linkage, such as a CPP and, independently, P. In some embodiments, the linker is linear and about 3 to about 100 (e.g., about 3 to about 20) atoms in length (not including branched atoms or substituents). In some embodiments, the linker provides a distance of about 1 Å to about 400 Å between the two groups it connects.

[0022] As used herein, "polypeptide" refers to a chain of at least two amino acids linked together by peptide bonds. There is no upper limit to the number of amino acids that can be included in a polypeptide. Furthermore, a polypeptide can include unnatural amino acids, amino acid analogs, or other synthetic molecules that can be incorporated into a polypeptide.

[0023] 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, the amino acid monomer may be trivalent if the monomer is further substituted. For example, a cysteine ​​monomer can independently form a peptide bond at the N-terminus and C-terminus, and can also form a disulfide bond.

[0024] As used herein, an "amino acid analog" or "analog" (e.g., an "arginine analog," "lysine analog," or "histidine analog") refers to a variant of an amino acid that retains at least one function of the amino acid, such as the ability to bind to an oligonucleotide via electrostatic interactions. Such variants may have elongated or shorter side chains (e.g., with one or more -CH2- groups that retain the ability to bind to an oligonucleotide through electrostatic interactions, or alternatively, modifications improve the ability to bind to an oligonucleotide via electrostatic interactions. For example, an arginine analog may include an additional methylene or ethylene between the backbone and the guanidine / guanidinium group. Other examples include amino acids with one or more additional substituents (e.g., Me, Et, halogen, thiol, methoxy, ethoxy, C1-haloalkyl, C2-haloalkyl, amine, guanidine, etc.). Amino acid analogs may be monovalent, divalent, or trivalent.

[0025] Throughout this specification, peptides and amino acid monomers are illustrated as charge-neutral species. It is understood that such species may carry a positive or negative charge depending on the conditions. For example, at pH 7, the N-terminus of an amino acid is protonated and bears a positive charge (-NH3 + ), and the C-terminus of the amino acid is deprotonated and carries a negative charge (-CO2 - ). Similarly, the side chains of certain amino acids can carry a positive or negative charge.

[0026] Each amino acid can be a natural or unnatural amino acid. The term "unnatural amino acid" refers to organic compounds that are analogs of the natural amino acids in that they have structures similar to the natural amino acids so as to mimic the structure and reactivity of the natural amino acids.

[0027] An unnatural amino acid may be a modified amino acid and / or an amino acid analog that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine. An unnatural amino acid may also be a D-isomer of a natural amino acid. Thus, as used herein, the term "amino acid" refers to natural and unnatural amino acids, as well as their analogs and derivatives. 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. An analog of an amino acid includes those that are structurally similar to, but not identical to, an amino acid, for example, due to modifications of the side chain or backbone on the amino acid. Such modifications may increase the hydrophobicity of the side chain, including elongation of the side chain with one or more hydrocarbons, or increasing the solvent accessible surface area (SASA, as described herein) of amino acids with aromatic rings in the side chain. For example, this can be done by conjugating a second aromatic ring or increasing the size of the aromatic ring. Derivatives of amino acids include natural and unnatural 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 with an alkylcarboxamidyl. These and other amino acids are listed in Table 1 along with their abbreviations as used herein. [Table 1-1] [Table 1-2]

[0028] "Alkyl" or "alkyl group" refers to the radical of a fully saturated, straight or branched hydrocarbon chain having from one to twelve carbon atoms and attached to the remainder of the molecule by a single bond. Alkyl containing any number of carbon atoms from 1 to 12 is included. Alkyl containing up to 12 carbon atoms is C1-C 12 Alkyl, alkyl containing up to 10 carbon atoms is C1-C 10 An alkyl having up to 6 carbon atoms is a C1-C6 alkyl, and an alkyl having up to 5 carbon atoms is a C1-C5 alkyl. C1-C5 alkyl includes C5 alkyl, C4 alkyl, C3 alkyl, C2 alkyl, and C1 alkyl (i.e., methyl). C1-C6 alkyl includes all of the moieties described above for C1-C5 alkyl, but also includes C6 alkyl. C1-C 10 Alkyl includes all of the moieties described above for C1-C5 alkyl and C1-C6 alkyl, but also includes C7, C8, C9 and C 10 Also includes alkyl. Similarly, C1-C 12 Alkyl includes all of the above moieties, but C 11 and C 12 Includes alkyl. C1-C 12 Non-limiting examples of alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless stated otherwise in the specification, alkyl groups can be optionally substituted.

[0029] "Alkylene" or "alkylene chain" refers to a fully saturated, straight or branched divalent hydrocarbon chain radical having 1 to 40 carbon atoms. C2-C 40Non-limiting examples of alkylene include ethylene, propylene, n-butylene, pentylene, etc. Unless stated otherwise in the specification, an alkylene chain can be optionally substituted as described herein.

[0030] "Alkenyl" or "alkenyl group" refers to a straight or branched hydrocarbon chain radical having from 2 to 12 carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the remainder 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 C2-C 12 Alkenyl, containing up to 10 carbon atoms, is C2-C 10 Alkenyl, an alkenyl group containing up to 6 carbon atoms is C2-C1 alkenyl, and an alkenyl containing up to 5 carbon atoms is C2-C5 alkenyl. C2-C5 alkenyl includes C5 alkenyl, C4 alkenyl, C3 alkenyl, and C2 alkenyl. C2-C6 alkenyl includes all of the moieties listed above for C2-C5 alkenyl, but also includes C6 alkenyl. C2-C 10 Alkenyl is C 2- Includes all of the moieties described above for C5 alkenyl and C2-C6 alkenyl, but includes C7, C8, C9 and C 10 Also includes alkenyl. Similarly, C2-C 12 Alkenyl includes all of the above moieties, but C 11 and C 12 Includes alkenyl. C2-C 12Non-limiting examples of alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), iso-propenyl, 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 stated otherwise in the specification, an alkyl group can be optionally substituted.

[0031] "Alkenyl" or "alkenyl chain" refers to a linear or branched divalent hydrocarbon chain radical having 2 to 40 carbon atoms and having one or more carbon-carbon double bonds. 40 Non-limiting examples of alkenylene include ethenylene (-CH=CH-), propenylene, butenylene, etc. Unless stated otherwise in the specification, an alkenylene chain can be optionally substituted.

[0032] "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 remainder 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 C2-C 12 Alkynyl, containing up to 10 carbon atoms, is C2-C 10 An alkynyl group containing up to 6 carbon atoms is C2-C6 alkynyl, and an alkynyl containing up to 5 carbon atoms is C2-C5 alkenyl. C2-C5 alkynyl includes C5 alkynyl, C4 alkynyl, C3 alkynyl, and C2 alkynyl. C2-C6 alkynyl includes all of the moieties listed above for C2-C5 alkynyl, but also includes C6 alkynyl. C2-C 10 Alkynyl includes all of the moieties listed above for C2-C5 alkynyl and C2-C6 alkynyl, but also includes C7, C8, C9 and C 10 Alkynyl is also included. Similarly, C2-C 12 Alkynyl includes all of the above moieties, but C 11 and C 12 Alkynyl is also included. C2-C 12 Non-limiting examples of alkenyls include ethynyl, propynyl, butynyl, pentynyl, etc. Unless stated otherwise in the specification, an alkyl group can be optionally substituted.

[0033] "Alkynylene" or "alkynylene chain" refers to a linear or branched divalent hydrocarbon chain radical having 2 to 40 carbon atoms and having one or more carbon-carbon triple bonds. 40 Non-limiting examples of alkynylene include ethynylene (-C≡C-), propargylene, etc. Unless stated otherwise in the specification, an alkynylene chain can be optionally substituted.

[0034] "Aryl" refers to a hydrocarbon ring system containing hydrogen, 6 to 40 carbon atoms, and at least one aromatic ring. For purposes of this disclosure, aryl can be a monovalent or divalent radical (without substituents), which can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, and can include fused or bridged ring systems. Aryl radicals include, but are not limited to, radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, and triphenylene. In some embodiments, the aryl radical can be divalent when used as or as part of a linker. Unless otherwise stated herein, aryl groups can be optionally substituted.

[0035] 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.

[0036] "Carbocyclyl", "carbocyclic", or "carbocycle" refers to a ring structure in which the atoms forming the ring are each carbon. Carbocyclic rings can contain 3 to 20 carbon atoms in the ring. Carbocyclic rings include aryl and cycloalkyl, as well as fully unsaturated, partially unsaturated, and fully saturated rings. In some embodiments, carbocyclyls can be divalent when used as or as part of a linker. Unless otherwise stated herein, carbocyclyl groups can be optionally substituted.

[0037] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon radical having 3 to 40 carbon atoms and at least one ring, which ring consists solely of carbon and hydrogen atoms and may include fused or bridged ring systems. For purposes of this disclosure, cycloalkyl can be a monovalent or divalent radical (without substituents). Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. In some embodiments, cycloalkyl radicals can be divalent when used as or as part of a linker. Unless otherwise stated herein, cycloalkyl groups can be optionally substituted.

[0038] "Cycloalkenyl" 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 double bonds, where the ring consists solely of carbon and hydrogen atoms and may include fused or bridged ring systems. For purposes of this disclosure, cycloalkenyl may be a monovalent or divalent radical (without substituents). Monocyclic cycloalkenyl radicals include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Polycyclic cycloalkenyl radicals include, for example, bicyclo[2.2.1]hept-2-enyl, and the like. In some embodiments, cycloalkenyl radicals may be divalent when used as or as part of a linker. Unless otherwise specified herein, cycloalkenyl groups may be optionally substituted.

[0039] "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, where the ring consists solely of carbon and hydrogen atoms and may include fused or bridged ring systems. For purposes of this disclosure, cycloalkynyl may be a monovalent or divalent radical (without substituents). Monocyclic cycloalkynyl radicals include, for example, cycloheptynyl, cyclooctynyl, and the like. In some embodiments, cycloalkynyl radicals may be divalent when used as or as part of a linker. Unless otherwise stated herein, cycloalkynyl groups may be optionally substituted.

[0040] "Heterocyclyl", "heterocyclic ring" or "heterocycle" refers to a stable 3- to 20-membered aromatic ring radical consisting of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. For purposes of this disclosure, a heterocyclyl radical can be a monovalent or divalent radical (without substituents). A heterocyclyl or heterocyclic ring includes heteroaryl, as defined below. Unless otherwise stated in the specification, a heterocyclyl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system and can include fused or bridged ring systems; the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals 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-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. In some embodiments, heterocyclyl radicals may be divalent when used as or as part of a linker. Unless stated otherwise in the specification, a heterocyclyl group may be optionally substituted.

[0041] "Heteroaryl" refers to a 5- to 20-membered ring system radical containing a hydrogen atom, 1-14 carbon atoms, 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For purposes of this invention, a heteroaryl radical can be a monovalent or divalent radical (containing no substituents), can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems; the nitrogen, carbon, or sulfur atoms in the heteroaryl radical can be optionally oxidized; and the nitrogen atom can be optionally quaternized. Examples include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthonofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indophenyl, iso ... Examples include, but are not limited to, dolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). In some embodiments, heteroaryl radicals when used as or as part of a linker can be divalent.Unless stated otherwise in the specification, a heteroaryl group may be optionally substituted.

[0042] As used herein, the term "ether" refers to a straight-chain or branched divalent radical moiety -[(CH2) m -O-(CH2) 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 specified herein, ethers may be optionally substituted.

[0043] The term "substituted" as used herein means any of the above groups (i.e., alkylene, alkenylene, alkynylene, aryl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, and / or ether) in which at least one hydrogen atom is replaced by a bond to a non-hydrogen atom, for example, but not limited to, halogen atoms such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl, alkoxy, and ester groups; sulfur atoms in groups such as thiol, thioalkyl, sulfone, sulfonyl, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl groups; and other heteroatoms of various other groups. "Substituted" also means any of the above groups in which one or more hydrogen atoms have been replaced with a higher order bond (e.g., a double bond or a triple bond) to a heteroatom, such as oxygen in groups such as oxo, carbonyl, carboxyl, and ester, and nitrogen in groups such as imine, oxime, hydrazone, and nitrile. For example, "substituted" means that one or more hydrogen atoms have been replaced with a bond such as -NR g R h , -NR g C(=O)Rh , -NR g C(=O)NR g R h , -NR g C(=O)OR h , -NR g SO2R h , -OC(=O)NR g R h , -OR g , -SR g , -SOR g , -SO2R g , -OSO2R g , -SO2OR g , =NSO2R g , and -SO2NR g R h "Substitution includes any of the above groups replaced with one or more hydrogen atoms by -C(=O)R. g , -C(=O)OR g , -C(=O)NR g R h , -CH2SO2R g , -CH2SO2NR g R h It is also meant to mean any of the above groups substituted with R g and R hare the same or different and are independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. "Substituted" further refers to any of the above groups in which one or more hydrogen atoms are replaced with a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl group. In addition, each of the aforementioned substituents may also be optionally substituted with one or more of the above substituents. Moreover, one of ordinary skill in the art will recognize that "substituted" encompasses instances in which any one or more atoms of the above groups are replaced by a substituent listed in this paragraph, which forms a covalent bond with the CPP, P, or L. For example, in certain embodiments, any of the above groups may 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 the group may be substituted at the second position with a thiol group that forms a disulfide bond with cysteine ​​(or an amino acid analog with a thiol group).

[0044] As used herein and in the concluding claims, a residue of a chemical species refers to the moiety that becomes a product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether it is actually derived from the chemical species. 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.

[0045] As used herein, [ka] The symbol (which may hereafter be referred to as an "adhesive bond") indicates a bond that is an adhesive bond between two chemical entities, one illustrating adhesion to the adhesive bond and the other not illustrating adhesion to the adhesive bond. For example, [ka] indicates that the chemical "XY" is attached to another chemical via an attachment bond point. Additionally, specific attachment points to chemicals not shown can be identified by inference. For example, the compound CH3-R 3 (In the formula, R 3 is H or [ka] ) is R 3 If "XY" is the adhesive bond point, R 3 It is assumed that the bond to CH3 is similar to the bond shown.

[0046] Unless otherwise stated, formulas with chemical bonds shown only with solid lines and no wedges or dashes contemplate each possible isomer, for example, each enantiomer, diastereomer, and mixtures of isomers, such as racemic or scalemic mixtures. The compounds described herein may contain one or more asymmetric centers and thus give rise to diastereomers and optical isomers. Unless otherwise stated, the compounds and compositions disclosed herein contain all possible diastereomers, as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and also their pharma- ceutically acceptable salts, mixtures of stereoisomers, and isolated specific stereoisomers. During the synthetic procedures used to prepare such compounds, or when using racemization or epimerization procedures known to those skilled in the art, the products obtained from such procedures may be mixtures of stereoisomers.

[0047] Many organic compounds exist in optically active forms, with the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule around its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) meaning that the compound is levorotatory. Compounds with a (+) or d prefix are dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are not superimposable mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein may have one or more chiral centers and therefore may exist as different enantiomeric forms. Optionally, a chiral carbon may be marked with an asterisk ( * ). When a bond to a chiral carbon is depicted as a straight line in a disclosed formula, it is understood that both the (R) and (S) configurations of the chiral carbon, and thus both enantiomers and mixtures thereof, are encompassed within the formula. As used in the art, when it is desired to specify the absolute configuration for a chiral carbon, one of the bonds to the chiral carbon may be depicted as a wedge (a bond to an atom above the plane) and the other as a series of short parallel lines (a bond to an atom below the plane). The Cahn-Ingold-Prelog system may be used to assign the (R) or (S) configuration to a chiral carbon.

[0048] The compounds described herein include atoms with both their natural isotopic abundance and non-natural isotopic abundance.The disclosed compounds may be isotopically labeled or isotopically substituted compounds, which are identical to those listed herein, but with respect to the fact that one or more atoms are replaced by atoms with atomic masses or mass numbers different from those usually found in nature.Examples of isotopes that can be incorporated into the compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F and 36 Compounds further comprising prodrugs thereof, and pharma- ceutically acceptable salts of the compounds or prodrugs that contain the aforementioned isotopes, and / or other isotopes of other atoms, are within the scope of the present invention. 3 Compounds into which radioactive isotopes such as H and C are incorporated are useful in drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Additionally, deuterium, i.e., 2 Substitution with heavier isotopes such as H may confer certain therapeutic benefits, such as increased half-life in vivo or reduced dosage requirements, resulting from greater metabolic stability, and therefore may be preferred in some cases. Isotopically labeled compounds and prodrugs thereof may generally be prepared by carrying out the following procedure by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0049] Disclosed are the components used to prepare the compositions disclosed herein and the compositions themselves used in the methods disclosed herein. When these and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even though specific reference to each of the various individual and collective combinations and permutations of these compounds may not be expressly disclosed. For example, when a particular compound is disclosed and discussed, and a number of modifications that can be made to a number of molecules including this compound are discussed, each and every combination and permutation of this compound, and possible modifications, are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and an example of a combination molecule A-D is disclosed, each is individually and collectively contemplated to mean the combination, even if each is not individually listed, and A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered to be disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, subgroups A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in the methods of making and using the compositions disclosed herein. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any particular embodiment or combination of embodiments of the methods disclosed herein.

[0050] The term "contacting" as used herein refers to bringing together a disclosed compound and a target (e.g., a cell, a target receptor, a transcription factor, or other biological entity) where the compound affects the activity of the target either directly, i.e., by interacting with the target itself, or indirectly, i.e., by interacting with another molecule, cofactor, factor, or protein on which the activity of the target depends.

[0051] As used herein, the terms "effective amount" and "amount effective" refer to an amount sufficient to achieve a desired result.

[0052] Reference will now be made in detail to certain aspects of the disclosed materials, compounds, compositions, articles and methods, examples of which are illustrated in the accompanying examples and drawings.

[0053] compound Disclosed are cell-penetrating peptides and compositions comprising them that can potentially provide a general vehicle for the cytosolic delivery of any peptide or protein cargo, as well as other biomolecules, including oligonucleotides. The disclosed peptides can have higher cytosolic delivery efficiency and in vivo stability than simple cell-penetrating peptides.

[0054] In certain aspects, disclosed herein are peptides that include 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, and at least one of the cell penetrating peptide motifs is 3-10 amino acid residues in length and has at least three arginine and / or lysine residues.

[0055] Unlike methods in which a CPP motif is inserted into each target protein, the disclosed compounds, compositions, and methods include engineered membrane translocation domains that can be genetically or synthetically fused to any target cargo of interest. Another strategy disclosed herein involves splitting the CPP motif into two halves and inserting them into two different regions of the membrane translocation domain, greatly improving cytosolic delivery efficiency.

[0056] In some embodiments, the compounds described herein can be used as plant activators. The term "plant activator" refers to a compound that activates the natural defense mechanism in a host plant, such as systemic acquired resistance (SAR) or hypersensitive response. The composition can be used as a plant activator for either healthy and unhealthy plants, or for plants in both healthy and unhealthy environments.

[0057] In some embodiments, the compounds described herein may be used as plant stimulants. The term "plant stimulant" as used herein refers to a compound or composition applied to a plant under conditions that enhance nutrient efficiency, stress resistance, and / or crop quality characteristics, regardless of its nutritional content. In particular, plant stimulants are used in plant culture to improve growth and development processes. The impact of stimulants on plants is not due to a direct involvement in the regulation of life processes, but rather an impact on metabolism in the broad sense of the term. They can stimulate the synthesis of natural hormones, sometimes increasing their activity, improve the uptake of minerals from the soil, and regulate root growth. In addition, they can cause an increase in resistance to adverse conditions (biotic or abiotic). The use of stimulants in plant culture increases the yield and often improves its quality at the same time. Stimulants can enhance the life processes occurring in plants without modifying the plant's natural behavior. The compounds and / or compositions described herein may be plant stimulants and therefore may be used as plant growth regulators, plant metabolic process regulators, plant physiological process regulators, substances that counteract the effects of biotic or abiotic stress in plants, and / or substances that confer multiple disease resistance to plants. The compositions may be used as plant stimulants for either healthy and unhealthy plants, or plants in both healthy and unhealthy environments.

[0058] Membrane translocation domain The membrane translocation domain portion of the disclosed peptides can be any membrane translocation domain, a peptide sequence capable of crossing a lipid bilayer, which has been modified to contain at least one cell-penetrating motif as described herein. In a preferred example, there are two or three cell-penetrating motifs in the membrane translocation domain. For example, at least one cell-penetrating peptide motif can be 3-10 amino acid residues long and have at least three arginine and / or lysine residues, e.g., 4, 5, or 6 arginine and / or lysine residues. Alternatively, at least one cell-penetrating peptide motif can be 3-10 amino acid residues long and have at least two arginine and / or lysine residues, and at least one other cell-penetrating peptide motif can be 2-8 amino acid residues long and have at least two hydrophobic residues. When there are two or more cell-penetrating peptide motifs, there can be two or more arginine and / or lysine residues within a 3-10 amino acid span, and there can be another cell-penetrating peptide motif with two or more hydrophobic residues within a 2-8 amino acid span. The cell-penetrating peptide motif can be present in any of the membrane translocation domains.

[0059] In some embodiments, the membrane translocation domain can be a plant membrane translocation domain. In some examples, the membrane translocation domain can be a human membrane translocation domain, such as fibronectin type III. In certain examples, the membrane translocation domain has at least 90%, at least 95%, or at least 97% sequence homology with SEQ ID NO: 118. In other examples, the membrane translocation domain is a human fibronectin type III having BC, DE, CD, and FG loops, and the cell penetrating peptide motif is in one or more of the BC, DE, CD, or FG loops, for example, the cell penetrating peptide motif is in two of the BC, DE, CD, or FG loops, particularly the BC and FG loops. These loops can be defined as having the following sequences: BC=AVTVR (SEQ ID NO: 31); CD=GGNSPVQ (SEQ ID NO: 32); DE=PGSK (SEQ ID NO: 33); FG=GRGDSPAS (SEQ ID NO: 34).

[0060] In other examples, the membrane translocation domain may be any stably folded protein that can be efficiently expressed in bacteria. Some further examples of membrane translocation domains are nanobody scaffolds, DARPin scaffolds, and CTPR proteins (consensus tetratricopeptide repeats, Acc. Chem. Res. 2021, 54, 4166-4177).

[0061] Cell-penetrating peptide motifs A cell penetrating peptide (CPP) motif can contain at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, or at least 6 amino acids, more specifically, 3-8, 3-6, 4-8, 4-6, or 6-8 amino acids. In most instances, the CPP motif is replaced with a membrane translocating domain, such that the resulting peptide has the same number of amino acids as the naturally occurring membrane translocating domain.

[0062] In some examples, at least 2, 3, 4, 5, 6, or 7 amino acids of the CPP motif are adjacent arginine residues. In preferred examples, there are 3, 4, or 5 adjacent arginine residues in the CPP motif. In other examples, the arginine residues are not adjacent in the CPP motif. Each amino acid in the CPP motif can be independently a natural or non-natural amino acid. When such adjacent arginine or lysine residues are CPP motifs, any additional CPP motifs, such as those with hydrophobic residues, are not required, although such hydrophobic CPP motifs may still be used. When the CPP motif contains two arginine residues, it is preferred that there is another CPP motif with at least two hydrophobic residues within 2-8 amino acids.

[0063] 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 a hydrophobic side chain. In some examples, the amino acids having 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-(naphthalen-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, tert-leucine, or nicotinoyllysine, each of which is optionally substituted with one or more substituents. In a specific example, each amino acid having a hydrophobic side chain is independently an amino acid having an aromatic side chain. In some embodiments, the amino acid having 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.Thus, in some examples, the amino acid having a hydrophobic side chain is phenylalanine, naphthylalanine, tryptophan, or an analog or derivative thereof, naphthylalanine or tryptophan, or an analog or derivative thereof.In other examples, the CPP motif further comprises at least one phenylalanine, phenylglycine, or histidine, or an analog or derivative thereof. [ka]

[0064] In some examples, the CPP motif can include any combination of at least three adjacent arginines, where at least two of the amino acids have a hydrophobic side chain selected from aryl or heteroaryl, where the aryl and heteroaryl are optionally substituted for a total number of amino acids in the CPP motif ranging from 5 to about 8 amino acids.

[0065] In some examples, the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops, and the CPP is in one or more of the BC, DE, CD, or FG loops. For example, the CPP motif is in two of the BC, DE, CD, or FG loops. In certain examples, the CPP motif is in the BC and any of the DE, CD, and FG loops, preferably in the BC and FG loops.

[0066] When more than one CPP motif is present, one CPP motif can be a segment of 3-10 amino acids having at least two arginine and / or lysine residues and the other can be a segment of 2-8 amino acids having at least two hydrophobic residues. For example, a membrane translocation domain can have more than one CPP, with at least one of the motifs being 2-8 amino acid residues and having at least two hydrophobic amino acid residues.

[0067] In this example, the membrane translocating domain can be human fibronectin type III having BC, DE, CD, and FG loops, the CPP motif can be in the BC loop, have 2-8 amino acid residues, and have at least two hydrophobic amino acid residues, the CPP motif can be in the FG loop, have 3-10 amino acid residues, and have at least three adjacent arginine and / or lysine residues, or the CPP motif can be in the FG loop, have 2-8 amino acid residues, and have at least two hydrophobic amino acid residues, and the CPP motif can be in the BC loop, have 3-10 amino acid residues, and have at least three adjacent arginine and / or lysine residues.

[0068] When the CPP motif contains 2-8 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. The CPP motif is preferably in the BC loop. More preferably, the CPP motif is WW, FW, WF, WYW, WWW, WWH, YWW, WYH, or YWH.

[0069] A CPP motif having 3-10 amino acid residues and at least three adjacent arginine and / or lysine residues may contain RRR, RRRR, RRRRR. It may also be any combination of arginine and lysine residues. If this CPP motif is in the FG loop, I may be 3-10 residues long and any combination of Arg and Lys (and possibly other non-acidic residues). A CPP motif (e.g., WWWRRRR) may alternatively be split such that some of the Arg / Lys residues are moved from the FG loop to the BC loop (e.g., WWWR...RRR, WWWRR...RR, WWWRRRR..., etc.); a CPP motif (e.g., WWWRRRR) may alternatively be split such that some of the 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 alternatively split such that either 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.).

[0070] In specific examples, the CPP motif comprises SEQ ID NO: 104, 105, 11, 112, 113, 114, 115, 116, or 117.

[0071] In some examples, the CPP motif may be or include any of the sequences listed in Table 2. In some examples, the cell penetrating peptide may be or include any of the reverse of the sequences listed in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0072] The chirality of the amino acids may be selected to improve cytosolic uptake efficiency. In some embodiments, at least two of the amino acids have opposite chirality. In some embodiments, at least two amino acids with opposite chirality may 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 may be adjacent to each other. In some embodiments, at least two of the amino acids have the same chirality. In some embodiments, at least two amino acids with the same chirality may 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 may be adjacent to at least two amino acids with the same chirality. Thus, in some embodiments, the adjacent amino acids of the cCPP have any of the following sequences: DL; LD; DLLD; LDDL; LDLLD; DLDDL; DLLDL; or LDDLD.

[0073] Cargo section The cargo moiety can be linked to the membrane translocation domain. The cargo moiety can be linked to an amino group (e.g., the N-terminus), a carboxylate group (e.g., the C-terminus), or a side chain of one or more amino acids in the membrane translocation domain.

[0074] When the cargo moiety is attached to the side chain of an amino acid in the membrane translocation domain, the membrane translocation domain contains an amino acid having a side chain with a suitable functional group for forming a covalent bond (conjugation) with the cargo, or a side chain that can be modified to provide a suitable functional group for forming a covalent bond with the cargo (e.g., via conjugation of a linker). In some embodiments, the amino acid on the membrane translocation domain with a suitable conjugation of the side chain of 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 embodiments, the cargo can be directly conjugated to the side chain of the amino acid (e.g., by forming a disulfide bond with the cysteine ​​residue, or an amide bond with the glutamic acid residue or the 2,3-diaminopropionic acid residue), or the cargo can be conjugated to the amino acid side chain via a linker (e.g., PEG).

[0075] In some embodiments, the cargo moiety may include a plant bioactive moiety. In some embodiments, the cargo moiety may further contain any cargo of interest, such as a linker moiety, a detectable moiety, or any combination thereof. In some examples, the cargo moiety may include one or more additional amino acids (e.g., K, UK, TRV); linkers (e.g., bifunctional linker LC-SMCC); coenzyme A; phosphocoumarin aminopropionic acid (pCAP); 8-amino-3,6-dioxaoctanoic acid (miniPEG); L-2,3-diaminopropionic acid (Dap or J); L-β-naphthylalanine; L-pipecolic 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 combinations thereof.

[0076] plant bioactive parts The cargo moiety may include a plant bioactive moiety. In some embodiments, a detectable moiety may be linked to the plant bioactive moiety. The plant bioactive moiety may be linked to the cell penetrating peptide moiety at an amino group, a carboxylate group, or any side chain of an amino acid of the cell penetrating peptide moiety (e.g., an amino group, a carboxylate group, or any side chain or amino acid of a CPP). In some examples, the plant bioactive moiety may be linked to a detectable moiety.

[0077] The term "plant bioactive moiety" refers to a compound that activates natural defense mechanisms in the host plant, such as systemic acquired resistance (SAR), or hypersensitive response, regardless of nutritional content, or that enhances nutritional efficiency, stress tolerance, and / or crop quality traits.

[0078] Non-limiting examples of plant bioactive moieties may include, but are not limited to, synthetically derived or naturally occurring flagellins and flagellin-related polypeptides (including those conserved within the genus Bacillus), thionin, harpin proteins or polypeptides, or harpin-like polypeptides, elongation factor Tu (EF-Tu), phytosulfokine (PSKα), root hair promoting polypeptides (RHPPs), hypersensitive response elicitor proteins or polypeptides, antifungal peptides, insect toxin peptides, antifreeze proteins, thermotolerant proteins, desiccation tolerance proteins, vitamin biosynthetic enzymes, bioherbicide peptides, or maize mitochondrial muteins, or any combination thereof. Each of the suitable synthetically derived or naturally occurring flagellins and flagellin-related polypeptides (including those conserved within the genus Bacillus), thionins, harpin proteins or polypeptides or harpin-like polypeptides, elongation factor Tu (EF-Tu), phytosulfokine (PSKα), root hair promoting polypeptides (RHPPs), and / or hypersensitive response elicitor proteins or polypeptides described herein or in International Application Publication Nos. WO2019 / 018768, WO2010 / 019442, WO1998 / 054214, WO2001 / 098501, and WO2013 / 102189 is incorporated herein by reference in its entirety. Plant bioactive moieties can be selected for their distinct modes of action and can be used individually or in combination with other polypeptides to address specific agricultural needs. They may be used in place of, or in addition to, commercial pesticides, biostimulants, supplemental bioactive agents, insecticidal compounds, or any combination thereof.

[0079] Flagellins and flagellin-related polypeptides derived from them have been reported to play functional roles mainly in innate immune responses in plants. These polypeptides are derived from highly conserved domains of eubacterial flagellins. Flagellins are the major components of bacterial flagella. The flagellin protein subunits that build bacterial eubacterial filaments can act as potent intracellular elicitors to initiate defense-related responses in various plant species.

[0080] "Flagellin" is a globular protein that arranges itself in a hollow cylinder to form a filament in bacterial flagellates. Flagellin is the major substituent of bacterial flagella and is present in flagellated bacteria. Plants can sense, combat infection, and initiate defense signaling against bacterial microorganisms by recognizing conserved epitopes, such as the 22 amino acid stretch (Flg22) located at the N-terminus of the full-length flagellin coding sequence. The elicitor activity of the Flg22 polypeptide is due to a conserved domain within the N-terminus of the flagellin protein (Felix et al., 1999). Plants can recognize bacterial flagellin through plant cell surface pattern recognition receptors (PRRs) known as flagellin sensors, which are leucine-rich repeat receptor kinases located in the plasma membrane and available at the plant cell surface. In plants, the best-characterized PRR is FLAGELLIN SENSING 2 (FLS2), which is highly conserved in both monocotyledonous and dicotyledonous plants.

[0081] Plant defensins are also characterized 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 provides a highly specialized role in protecting plants against microbial pathogens (Nawrt et al., “Plant antimicrobial peptides,” Folia Microbiology 59:181-196, 2014).

[0082] Thionin is a cystine-rich plant AMP classified in the defensin family, which usually contains 45-48 amino acid residues, 6-8 of which are cysteines that form 3-4 disulfide bonds in higher plants. Thionin has been found to be present in both monocotyledonous and dicotyledonous plants, and its expression can be induced by infection with various microorganisms (Tam et.al., “Antimicrobial peptides from plants,” Pharmaceuticals 8:711-757, 2015). Specific amino acids of thionin, such as Lys1 and Tyr13, which are highly conserved, have been found to be important for the functional toxicity of these AMPs.

[0083] Harpins and harpin-like proteins are similar to flagellins or flagellin-related polypeptides. Harpins comprise a group of bacterially derived elicitors derived from larger precursor proteins. Harpins are essential for triggering the hypersensitive response (HR) when infiltrated into the intercellular space or apoplast of plant cells (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). Application of a remote harpin-like bioactive priming polypeptide(s) to plants provides an alternative conduit for protecting plants from disease and insect pressure. Harpins utilize a type III secretion system that allows transport of proteins across the lipid bilayer that constitutes the plant plasma membrane. Binding of harpin to the surface of the plasma membrane may trigger an innate immune response that is similar to the immune response triggered by pathogen-associated molecular patterns (PAMPs) and is known to activate PAMP-triggered immunity (Engelardt et al., “Separable roles of the Pseudomonas syringae pv. phaseoliola accessory protein HrpZ1 in ion-conducting pore formation and activation of plant immunity,” The Plant Journal 57:706-717, 2009).Mutational analyses of polypeptides derived from harpin-like HpaG showed that 12 amino acid residues between Leu-39 and Leu50 of the original 133 amino acid harpin elicitor precursor protein are important for hypersensitive (HR) and subsequent induction of innate immune responses in tobacco (Kim et al., “Mutational analyses 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 indicates that a specific amino acid region of harpin (similar to other AMPs) is involved in the elicited response. Harpins such as HpaG-like may be used to enhance resistance not only to plant pathogens but also to insects (Choi et al., “Harpins, multifunctional proteins secreted by gram-negative plant pathogenic bacteria,” Molecular Plant Microbe Interactions 26:1115-1122, 2013). Harpins have been used to induce disease resistance in plants and protect plants from colonization and feeding by phloem-feeding insects such as aphids (Zhang et al., “Harpin-induced expression and transgenic overexpression of phloem protein gene At.PP2A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae,” BMC Plant Biology 11:1-11, 2011).

[0084] In some embodiments, harpin proteins or polypeptides or harpin-like polypeptides may include, but are not limited to, homologs of Erwinia amylovora HrpN, including those from Erwinia, Pantoea, and Pectobacterium species. Examples of such homologs include those from Genbank accession numbers AAC31644 (Erwinia amylovora); AAQ21220, AAQ17045, CAE25423, CAE25424, CAE25425, and CAF74881 (Erwinia pyrifoliae); CAC20124, Q47278, Q47279, and AAY17519 (Erwinia chrysanthemi); CAE25422 (Erwinia strain JP557); AAG01466 (Pantoea stewartii); AAF76342 (Pantoea agglomerans); ABZ05760, ABI15988, ABI15989, ABI15990, ABI15991, ABI15992, ABI15996, ABK80762, ABD04037, ABI15994, ABD04035, ABD04036, AAY17521, AAX38231, ABI15995, AAQ73910, and CAL69276 (Pectobacterium carotovorum); YP_050198, AAS20361, and CAE45180 (Pectobacterium atrosepticum); and ABD22989 (Pectobacterium betavasculorum), each of which is incorporated herein by reference in its entirety.

[0085] Another group of harpin proteins or polypeptides or harpin-like polypeptides may include, but are not limited to, homologs of Erwinia amylovara HrpW and Pseudomonas syringae HrpW, including those from species of Erwinia, Pseudomonas, Xanthomonas, Acidovorax, and Pectobacterium. Examples of such homologs include Genbank accession numbers U94513, CAA74158, AAC04849, and AAF63402 (Erwinia amylovara); AAQ 17046 (Erwinia pyrifoliae); YP OO 1906489 (Erwinia tasmaniensis); YP_050207 (Pectobacterium atrosepticum); AF037983 (Pseudomonas syringae pv. tomato); AAO50075 (Pseudomonas syringae pv. phaseoliola), AAL84244 (Pseudomonas syringae pv.(maculicola) AAX58453,AAX58541,AAX58589,AAT96311,AAX58497,AAX58579,AAX58449,AAX58485,AAX58563,AAX58581,AAX58575,AAX58569,AAX58567,AAX58505,AAX5 8591,AAX58503,AAX58507,AAX58509,AAX58469,AAX58441,AAX58543,AAX584 95,AAX58549,AAX58593,AAX58511,AAX58519,AAT96270,AAX58447,AAX58571 (Pseudomonas viridiflava); ABA47299 and BAG24117 (Pseudomonas cichorii); CAH57075 (Pseudomonas avellanae); BAE80274 and BAE80242 (Acidovorax avenae), and AAM37767 (Xanthomonas axonpodis pv. citri), each of which is incorporated herein by reference in its entirety.

[0086] Yet another group of harpin proteins or polypeptides or harpin-like polypeptides may include, but are not limited to, homologs of Pseudomonas syringae HrpZ, including those from other species of Pseudomonas. Examples of such homologs include those listed under Genbank accession numbers P35674, AAB00127, ABL01505, AAQ92359, BAD20880, BAD20876, BAD20892, BAD20884, BAD20928, BAD20936, BAD20932, BAD20924, BAD20856, BAD20864, ​​BAD20860, BAD20848, BAD208 44,BAD20836,BAD20840,BAD20824,BAD20842,BAD20820,BAD20916,BAD20872,BAC81526,087653,BAA74 798,BAD20904,AAB86735,BAD20912,BAD20908,ABL01504,BAB40655,ABO26225,ABO26228(Pseudomonas syringae pv.); BAD20868 (Pseudomonas ficuserectae); AAX52452, AAT96159, AAX52266, AAX52396, AAT96322, AAT96281, AAX52272, AAX52306, AAX52270, AAX52402, AAX52276, AAX52318, AAX52262, and AAT96361 (Pseudomonas viridiflava); CAJ76697 (Pseudomonas avellanae); YP OO1 185537 (Pseudomonas mendocina); and ABA47309 and BAG24128 (Pseudomonas cichorii), each of which is incorporated herein by reference in its entirety. An additional group of harpin proteins or polypeptides or harpin-like polypeptides is found in Xanthomonas campestris HreX (see U.S. Patent No. 6,960,705 to Wei et al.).This may include, but is not limited to, homologs of Xanthomonas campestris (which are incorporated herein by reference in their entireties), including homologs from other species of Xanthomonas. 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 axonopodis), each of which is incorporated herein by reference in its entirety.

[0087] In some embodiments, the harpin protein or polypeptide or harpin-like polypeptide is a fragment or combination of fragments (i.e., a fusion protein) of one of the harpin proteins mentioned above. In some embodiments, the harpin fragment or fusion protein includes a fragment that induces a hypersensitive response. In another embodiment, the harpin fragment or fusion protein includes a fragment that induces a hypersensitive response. Suitable harpin cross sections include, for example, two structural units, a stable α-helical unit having a length of 12 or more amino acids; a hydrophilic, acidic unit having a length of 12 or more amino acids that may be in a beta form, a beta turn, or a disordered form. The fragment may also be characterized by an acidic pi value, preferably of about 5 or less. The cross section may contain any number of amino acids, for example, from about 25 to about 60, or from about 28 to about 40 amino acids.

[0088] Examples of suitable harpin fragments or fusion proteins are identified 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 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.

[0089] A fragment or fusion protein currently commercially available from Plant Health Care Inc. is characterized by the amino acid sequence of SEQ ID NO: 129, as follows: MSLNTSGLGASTMQISIGGAGGNNGLLGTHMPGTSSSPGLFQSGGDNGLGGHNANSALGQQPIDRQTIEQMAQLLAELLKSLLDSGEKLGDNFGASADSASGTGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDSGPAKDGNAGANDPSKNDPSKSQGPQSANKTGNVDDANNQDPMQALMQLLEDLVKLLKAALHMQQ PGGNDKGNGVGGDSGQNDDSTSGTDSTSDSSDPMQQLLKMFSEIMQSLFGDEQDGTDSTSGSRFTRTGIGMKAGIQALNDIGTHSDSSTRSFVNKGDRAMAKEIGQFMDQYPEVFGKPQYQKGPGQEVKTDDKSWAKALSKPDDDGMTPASMEQFNKAKGMIKSAMAGDTGNGNLQARGAGGSSLGIDAMMAGDAINMALGKLGAA

[0090] The harpin fragment or fusion protein of SEQ ID NO:129 is encoded by the nucleotide sequence of SEQ ID NO:130, as follows:

[0091] Elongation factor Tu is an abundant protein found in bacteria that acts as a pathogen-associated molecular pattern (PAMP) and initiates signaling cascades involved in plant disease resistance and innate immunity of plants against microbial pathogenic organisms. Interestingly, some EF-Tu polypeptides have also been found to be present in plants. The first 18 amino acid residues at the N-terminus of EF-Tu from Escherichia coli (called elf18) are 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). Polypeptides derived from E. coli EF-Tu are recognized by the plant cell surface-localized receptor EF-Tu receptor (EFR) (Zipfel et al., 2006). EF-Tu binding and EFR activation follow a similar mode of action compared to that of 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 USA113:11034-9, 2016).

[0092] Phytosulfokines (PSKs) belong to a group of sulfated plant polypeptides that are ubiquitous and encoded by precursor genes that are highly conserved in higher plants (Sauter M., “Phytosulfokine peptide signaling,” Journal of Experimental Biology 66:1-9, 2015). PSK genes are encoded by a family of small genes that are present in both monocotyledonous and dicotyledonous plants and encode PSK polypeptide(s) that may be active as either pentapeptides or C-terminally truncated tetrapeptides (Lorbiecke R, Sauter M, “Comparative analysis of PSK peptide growth factor precursor homologs,” Plant Science 163:348-357, 2002).

[0093] Phytosulfokine proteins are targeted to the secretory pathway in plants by conserved signal polypeptides (Lorbiecke R, Sauter M, “Comparative analysis of PSK peptide growth factor precursor homologs,” Plant Science 163:348-357, 2002). Phytosulfokines (PSKs) function as sulfated growth factors with biostimulant activity and are involved in the development of root and shoot apical meristems, growth regulation, and the control of reproductive processes. PSK has also been reported to initiate cell proliferation, differentiation of quiescent tissues, and to be involved in the formation and stimulation of tracheary element differentiation (Matsubayashi et al., "The endogenous sulfated pentapeptide phytosulfokine-α stimulates 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 the regulation of root and hypocotyl elongation in Arabidopsis seedlings (Kutschmar et al., "PSK-α promotes root growth in Arabidopsis," New Phytologist 181:820-831, 2009).

[0094] Root hair promoting polypeptide (RHPP) is a 12 amino acid fragment derived from the soybean Kunitz trypsin inhibitor (KTI) protein and is expressed in Bacillus circulans HA. 12It was detected in soybean meal subjected to degradation using alkaline protease derived from soybean (Matsumiya Y. and Kubo M. “Soybean and Nutrition, Chapter 11: Soybean Peptide: Novel plant growth promoting peptide from soybean,” Agriculture and Biological Sciences, Sheny HE (editor), pgs. 215-230, 2011). When applied to soybean roots, RHPP was shown to accumulate in the roots and promote root growth in Brassica through stimulation of cell division and root hair differentiation.

[0095] Detectable Part Detectable moiety can include any detectable label.Suitable detectable label examples include, but are not limited to, UV-Vis label, near infrared label, luminescent group, phosphorescent group, magnetic spin resonance label, photosensitizer, photocleavable moiety, chelating center, heavy atom, radioisotope, isotopically detectable spin resonance label, paramagnetic moiety, chromophore, or any combination thereof.In some embodiments, label can be detected without adding additional reagent.

[0096] In some embodiments, the detectable moiety is a biocompatible detectable moiety such that the compound may be suitable for use in a variety of biological applications. As used herein, "biocompatible" and "biologically compatible" generally refer to compounds that, together with any of their metabolic or decomposition products, are generally non-toxic to cells and tissues and do not produce any significant adverse effects on cells and tissues when the cells and tissues are incubated (e.g., cultured) in their presence.

[0097] The detectable moiety can contain 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, perylenediimine, pyrene, azo dyes, xanthene dyes, boron dipyrromethenes, aza-boron dipyrromethenes, cyanine dyes, metal-ligand complexes such as bipyridines, bipyridyls, phenanthrolines, coumarins, and acetylacetonates of ruthenium and iridium, acridines, oxazine derivatives such as benzophenoxazines, aza-annulenes, squaraines, luminescent nanoparticles such as 8-hydroxyquinolines, polymethines, quantum dots, nanocrystals, carbostyrils, terbium complexes, inorganic phosphorus, ionophores such as crown ether-related dyes or derivatized dyes, or combinations thereof. Specific examples of suitable luminophores include, but are not limited to, Pd(II) octaethylporphyrin; Pt(II)-octaethylporphyrin; Pd(II) tetraphenylporphyrin; Pt(II) tetraphenylporphyrin; Pd(II) meso-tetraphenylporphyrin tetrabenzoporphin; Pt(II) meso-tetraphenylmethrylbenzoporphyrin; Pd(II) octaethylporphyrin ketone; Pt(II) octaethylporphyrin ketone; Pd(II) meso-tetra(pentafluorophenyl)porphyrin; Pt(II) meso-tetra(pentafluorophenyl)porphyrin; Ru(II) tris(4,7 -diphenyl-1,10-phenanthroline)(Ru(dpp)3);Ru(II) tris(1,10-phenanthroline)(Ru(phen)3);Tris(2,2'-bipyridine)ruthenium(II) chloride hexahydrate(Ru(bpy)3);Erythrosin 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;Macrolex fluorescent red;Macrorex fluorescent yellow;Texas red;Rhodamine B;Rhodamine 6G;Sulfur rhodamine;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;Calmagite;Bromo-xylenol;Phenol red;Neutral red;Nitrazine;3,4,5,6-tetrabromophenolphthalein;Congo red;Fluorescein;Eosin;2',7'-dichlorofluorescein fluorescein;5(6)-carboxy-fluorescein;carboxynaphthofluorescein;8-hydroxypyrene-1,3,6-trisulfonic acid;semi-naphthorhodafluor;semi-naphthofluorescein;tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride;(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) tetraphenylborate;platinum(II) octaethylporphyrin;dialkylcarbocyanines;dioctadecylcyclooxacarbocyanine;fluorenylmethyloxycarbonyl chloride;7-amino-4-methylcoumarin (Amc);green fluorescent protein (GFP);and derivatives or combinations thereof.;

[0098] In some examples, the detectable moiety may include rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), naphthofluorescein (NF), or derivatives or combinations thereof.

[0099] The detectable moiety can be attached to the cell-penetrating peptide moiety at an amino group, a carboxylate group, or the side chain of any amino acid of the cell-penetrating peptide moiety (e.g., an amino group, a carboxylate group, or the side chain of any amino acid in a CPP).

[0100] Linker In various embodiments, the linker is covalently attached to an amino acid on the membrane translocation domain. The linker can be any moiety that conjugates the membrane translocation domain to the cargo moiety. In some embodiments, the linker can be an amino acid. In other embodiments, the precursor to the linker can be any suitable molecule that can form two or more bonds with the amino acids in the membrane translocation domain and the cargo moiety. Thus, in various embodiments, the precursor to the linker has two or more functional groups, each of which can form a covalent bond to the membrane translocation domain and the cargo moiety. For example, the linker can be covalently attached to the N-terminus, C-terminus, or side chain, or combinations thereof, of any amino acid in the membrane translocation domain. In certain embodiments, the linker forms a covalent bond between the membrane translocation domain and the cargo moiety. In some embodiments, the linker can be an unstructured polypeptide sequence. In some embodiments, when the linker is an unstructured polypeptide sequence, it allows the membrane translocation domain, linker, and cargo conjugate to be produced recombinantly.

[0101] 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 may be optionally substituted as defined above. Non-limiting examples of linkers include polyethylene glycol, optionally conjugated to a lysine residue.

[0102] In some embodiments, the linker may be 0 to 1000 amino acids in length. In some embodiments, the linker may be designed to be a repeat of Gly-Gly-Ser. In some embodiments, the linker sequence may be a linker described in Adv Drug Deliv Rev. 2013 October 15;65(10):1357-1369. For example, (G) such as SEQ ID NO: 131 GGGGS and SEQ ID NO: 132 (GGGGS)3. n(n = 1 to 10), or SEQ ID NO: 131 (GGGGS) n (n=1 to 4) A flexible linker sequence defined by SEQ ID NO: 133 A (EAAAK) n A(n=2~5), or (XP) n (n=5 to 20) (wherein X is any amino acid, preferably SEQ ID NO: 134 A(EAAAK)4ALEA(EAAAK)4A, SEQ ID NO: 135 AEAAKEAAAKA, SEQ ID NO: 136 PAPAP, (Ala-Pro) n rigid linker sequences defined by a cleavable linker sequence such as a disulfide or protease sensitive sequence (e.g., SEQ ID NO: 137 VSQTSKLTR↓AETVFPDV, etc. (n=5-17), which represent Ala, Lys, or Glu); 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: 143 AGNRVRR↓SVG, SEQ ID NO: 144 RRRRRRR↓R↓R d , or SEQ ID NO: 145 GFLG↓ e (where a Protease-sensitive cleavage sites are indicated by “↓”; b factor XIa / factor FVIIa sensitive cleavage; c Matrix metalloproteinase-1 sensitive cleavage sequence (an example is provided herein); d HIV PR (HIV-1 protease), NS3 protease (HCV protease), and factor Xa-sensitive cleavage, respectively; e Furin-sensitive cleavage; and f Cathepsin B-sensitive cleavage).

[0103] In some embodiments, the linker comprises the N-terminus or C-terminus of an amino acid on the CPP motif, or the side chain of glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain bearing an amino group). In specific embodiments, the linker forms a bond with the side chain of glutamine on the CPP motif. In other specific embodiments, the linker described herein has the structure of L-1 or L-2: [ka] and [In the formula, AA s is the side chain or terminus of an amino acid on a peptide or staple; AA c is the side chain or terminus of an amino acid of the cCPP; p is an integer from 0 to 10); and q is an integer from 1 to 50.

[0104] In some embodiments, the linker can release the cargo moiety from the membrane translocation domain after the polypeptide conjugate enters the cytosol of a 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 cytosolic uptake of the polypeptide conjugate, thereby releasing the cargo moiety. Non-limiting examples of physiologically cleavable linking groups include carbonate, thiocarbonate, thioether, thioester, disulfide, sulfoxide, hydrazine, protease-cleavable dipeptide linkers, and the like.

[0105] For example, in some embodiments, the linker is covalently attached to the membrane translocation domain via a disulfide bond with, for example, a side chain of a cysteine ​​or cysteine ​​analog located in the membrane translocation domain or cargo moiety. In some embodiments, the disulfide bond is formed between a thiol group on the precursor of the linker and a side chain of a cysteine ​​or amino acid analog having a thiol group on the peptide, and the bond to the hydrogen in each of the thiol groups is replaced by a bond to a sulfur atom. Non-limiting examples of amino acid analogs having a thiol group that can be used with the polypeptide conjugates disclosed herein are discussed above.

[0106] Manufacturing method The compounds described herein can be prepared by various methods known to those skilled in the art of organic synthesis, or variations thereof that will be appreciated by those skilled in the art.The compounds described herein can be prepared from readily available starting materials.Optimal reaction conditions may vary depending on the particular reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0107] Variations of the compounds described herein include the addition, deletion, or movement of various components described for each compound. Similarly, the chirality of the molecule can be altered if there is one or more chiral centers in the molecule. Furthermore, the synthesis of the compounds can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of suitable protecting groups, can be determined by those skilled in the art. The chemistry of protecting groups is described, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0108] Starting materials and reagents used in preparing the disclosed compounds and compositions may be purchased from 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 They are available from commercial manufacturers such as Ingelheim (Ingelheim, Germany) or are prepared by methods known to those skilled in the art following procedures described 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 Supplementals (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 disclosed herein, such as the pharmaceutical carriers, can be obtained from commercial sources.

[0109] The reactions to produce the compounds described herein can be carried out in a solvent that can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive to the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) at which the reaction is carried out. The reaction can be carried out in one solvent or a mixture of multiple solvents. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) It can be monitored by infrared spectroscopy, spectrophotometry (eg, UV-visible), or mass spectrometry, or chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0110] The disclosed compounds can be prepared by expression and purification in the same way as any other protein. See Chen, K., & Pei, D. (2020). Engineering cell-penetrating proteins by insertion of cell-penetrating motifs into surface loops. ACS Chemical Biology, 15(9), 2568-2576, which is incorporated by reference in its entirety for teaching how to prepare proteins. Other methods for preparing the compositions of the present disclosure include solid-phase peptide synthesis in which the α-N-terminus of the amino acid is protected by an acid or base protecting group. Such protecting groups should have the properties of being stable to the conditions of peptide bond formation and easily removable without disruption of the growing peptide chain or racemization of any chiral centers contained therein. Suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, etc. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. Other preferred side chain protecting groups are: lysine and arginine, side chain amino groups such as 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; tyrosine, benzyl, o-bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl, and acetyl (Ac); serine, t-butyl, benzyl, and tetrahydropyranyl; histidine, trityl, benzyl, Cbz, p-toluenesulfonyl, and 2,4-dinitrophenyl; tryptophan formyl; aspartic acid and glutamic acid, benzyl and t-butyl; and cysteine, triphenylmethyl (trityl).In the solid phase peptide synthesis method, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are those materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as insoluble in the media used. The solid support for the synthesis of α-C-terminal carboxypeptides is 4-hydroxymethylphenoxymethyl-copoly(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) mediated coupling at temperatures between 10°C and 50°C in a solvent such as dichloromethane or DMF with or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl) 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, before coupling with the α-C-terminal amino acid as described above. In one method for coupling to the deprotected 4(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) were prepared in DMF. The coupling of successive protected amino acids may be performed in an automated polypeptide synthesizer. In one example, the α-N-terminus of an amino acid of the growing peptide chain is protected with 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 in approximately 3-fold molar excess and coupling is preferably carried out in DMF.

[0111] The coupling agents can be O-benzotriazol-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, either sequentially or in a single operation. Removal and deprotection of the polypeptide can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thianizole, water, ethanedithiol, and trifluoroacetic acid. If 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 transamination. The protected peptide can be purified at this point or proceed directly to the next step. Removal of the side chain protecting groups can be accomplished using the cleavage cocktail described above. The fully deprotected peptide may be purified by a series of chromatographic steps using 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 carboxymethylcellulose; partition chromatography, (e.g., Sephadex G-25, LH-20, or countercurrent distribution); high performance liquid chromatography (HPLC), especially reversed-phase HPLC on octyl- or octadecylsilyl-silica bonded phase column packings.

[0112] How to use Also 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 a plant cell with a peptide described herein.Also provided are methods of delivering a plant activator into a plant cell, comprising contacting a plant cell with a peptide described herein.Also provided are methods of delivering a plant bioactive moiety into a plant cell, comprising contacting a plant cell with a peptide described herein.

[0113] Also provided is a method for delivering a plant stimulant into a plant, comprising contacting the plant with the peptides described herein.Also provided is a method for delivering a plant activator into a plant, comprising contacting the plant with the peptides described herein.Also provided is a method for delivering a plant bioactive part into a plant, comprising contacting the plant with the peptides described herein.

[0114] Methods for treating a plant having a disease caused by a pathogenic agent are also described. The methods can include contacting the plant with an effective amount of a compound and / or composition described herein.

[0115] In some aspects, the compounds or compositions described herein are used to protect plants from biotic stress caused by living organisms such as fungi, bacteria, nematodes, insects, mites, and animals; to stimulate seeds during germination; to protect plants from abiotic stress caused by physical or chemical stressors of non-living origin due to the presence of harmful chemicals including salts, limited access to water, sunburn, freeze damage, wind damage, nutrient deficiencies, or improper cultural practices such as overwatering or planting too deeply; to enhance plant growth, yield, health, longevity, productivity, and / or vigor; and / or to confer multiple disease resistance to plants.

[0116] Also provided are methods of protecting plants from biotic stress; stimulating seeds during germination; protecting plants from abiotic stress; enhancing plant growth, yield, health, longevity, productivity, and / or vigor; conferring multiple disease resistances to a plant; or any combination thereof. The methods may include contacting a plant cell with a peptide described herein.

[0117] The compounds or compositions described herein confer resistance to various pathogens. In some embodiments, the compounds or compositions can be used as plant stimulants for plants with diseases caused by pathogens. The pathogens can include fungi, viruses, bacteria, mycoplasma, spiropramine or viroids. Exemplary pathogens can include fungi such as Erisyphe polygoni, Phytophthora capsicci, Verticillium dahliae and other Verticillium species, powdery mildew, and Fusarium species; bacteria such as Pseudomonas syringae py. 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 infestants of tomato, Plasmopara viticola, Pseudomonas tomato, Phytophthoraparasiticavar.nicotiniae, Peronospora tabacina, Cercospora nicotianae, Phytophthora parasitica var.nicotiniae, Peronospora tabacina, Cercospora nicotianae, Pseudomonas tabaci, Erysiphe graminis, Phytophora medicaginis, P. megasperma, Pyricularia oryzae, Helminthosporium Helminthosporium leaf blight such as oryzae, Cochliobolus miyabeanus, Gibberella Seedling blight such as fujikuroi, seedling damping-off such as Rhizopus oryzae, sheath blight such as Rhizoctonia solani, Puccinia coronata, Erysiphe graminis, Rhynchsporium secalis,Cochliobolus sativus, Helminthosporium gramineum, Pyrenophora gramineum, Pyrenophra teres, Tilletia caries, Ustilago nuda, Leptosphaeria nodorum, Septoria nodorum, Puccinia striiformis, Typhula incamata, Pseudocercosporella herpotrichoides, Calonectria graminicola, 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, black spot disease,and other diseases of cereals, grains, beets, legumes, pome fruits, stone fruits, berries, citrus fruits, oil plants, cucumber plants, fiber plants, lauraceae, ornamental plants, and also oilseed rape, sunflowers, carrots, peppers, strawberries, melons, kiwi fruit, onions, leeks, sweet potatoes, figs, plums, asparagus, persimmons, soybeans, adzuki beans, watermelons, chrysanthemums, spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, paprika, tea, wheat, hemp, rye, oats, rice, sorghum, sugar beet, fodder beet, phosphorus Plants that may be used include goats, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries, beans, lentils, peas, soybeans, rapeseed, mustard, poppies, olives, sunflowers, coconuts, castor beans, cocoa beans, groundnuts, cucumbers, mallows, melons, cotton, flax, cannabis, jute, oranges, lemons, grapefruit, mandarins, avocados, cinnamon, camphor, corn, tobacco, nuts, coffee, sugarcane, tea, vines, hops, bananas, natural rubber plants, flowers, shrubs, deciduous and coniferous trees, etc.

[0118] The compounds or compositions described herein may be effective against a wide variety of insects. The European corn borer is a major pest of corn (dent and sweet corn), but also feeds on over 200 plant species, including beans, wax beans, lima beans, soybeans, peppers, potatoes, tomatoes, and many weed species. In addition, pests that feed on insect larvae and damage a wide variety of vegetable crops include, but are not limited to, the beet armyworm, the nettle looper, the tobacco budworm larvae, the fall armyworm, the diamondback moth, the cabbage fly larvae, the onion fly, the seed fly, the pyralidae larvae (melon worm), the pepper maggot, and the tomato pinworm.

[0119] With regard to the use of compounds or compositions to enhance plant growth, various forms of plant growth enhancement or promotion can be achieved. This can occur as early as when plant growth is initiated from seed, or later in the growth stage. For example, plant growth according to the present invention includes increased yield, increased seed volume produced, increased seed germination rate, increased plant size, increased biomass, larger fruit, earlier fruit coloring, and earlier fruit and plant maturation. For example, early germination and early maturation allow crops to grow in areas where the growing season is short and growth there may be hindered. Increased seed germination percentage improves crop height and uses seeds more efficiently. Higher yield, larger size, and enhanced biomass production allow for greater revenue generation from a given plot of land. In some embodiments, the compounds or compositions can be used to promote early flowering.

[0120] As used herein, "health of a plant" or "plant health" refers to the condition of a plant and / or its products as determined by several aspects alone or in combination with each other, such as, for example, increased yield, vigor, quality, and resistance to abiotic and / or biotic stresses.

[0121] Plants that are attacked by fungi or pesticides often produce less biomass, and therefore yield is reduced, compared to plants that are subjected to therapeutic or preventive treatment against pathogenic fungi or any other related pests and can grow without being damaged by biotic stress factors.However, applying the compounds and / or compositions described herein enhances plant health even in the absence of biotic stress.Applying the compounds and / or compositions described herein to plants and / or cultivation areas can also be carried out in the absence of pest pressure on the plant.

[0122] According to the present invention, "increasing the yield of a plant" means that the yield of a product of the plant is increased by a measurable amount over the yield of the same product of the plant produced under the same conditions, but the compound and / or composition is not applied to the plant and / or the cultivation area. In one embodiment, the term "yield" refers to fruits, as appropriate, as well as vegetables, nuts, grains, and seeds.

[0123] "Crops" and "fruits" are understood to be any plant products which are further utilized after harvest, such as fruits, vegetables, nuts, grains, seeds, timber (e.g. in the case of silvicultural plants), flowers (e.g. in the case of horticultural and ornamental plants), etc., in the appropriate sense.

[0124] Increased plant yield can be characterized by the following non-limiting characteristics: increased plant weight; increased biomass, such as increased total fresh weight (FW) and / or increased total dry weight (DW), increased number of flowers per plant; increased grain and / or fruit yield; more chillies or side shoots (branching); larger leaves; enhanced shoot growth; increased protein content; increased oil content; increased starch content; increased pigment content; increased chlorophyll content; and any combination thereof.

[0125] Chlorophyll content is positively correlated with the photosynthetic rate of plants, therefore the higher the chlorophyll content, the higher the plant yield.

[0126] Increasing plant yield may involve improved vigor. Vigor is manifested in several aspects including the general visual appearance of the plant. Improved vigor may be characterized, among others, by: improved plant vigor; improved plant growth; improved plant development; improved visual appearance; improved plant height (less plant locus / burrowing); improved trichomes; enhanced root growth and / or a more developed root system; enhanced nodulation, especially rhizobia formation; larger leaf blades; larger size; increased plant height; increased chilli number; increased number of side shoots; increased number of flowers per plant; enhanced shoot growth; enhanced root growth (extensive root system); enhanced photosynthetic activity; increased pigment content; earlier flowering; earlier fruiting; earlier and improved germination. improved germination; earlier grain maturity; fewer unproductive chillers; fewer dead basal leaves; fewer inputs required (such as fertilizer or water); greener leaves; full maturity under shortened growing season; less fertilizer required; less seed sowing required; earlier harvest; faster and more uniform maturation; longer shelf life; longer panicles; delayed senescence; stronger and / or more productive chillers; improved extractability of ingredients; improved seed quality (to be sown the following season for seed production); reduced inhibition of ethylene production and / or its acceptance by plants; and any combination thereof.

[0127] The enhancement of the photosynthetic activity of a plant may be based on an increase in the stomatal conductance and / or an increase in the rate of CO2 assimilation of the plant.

[0128] Increasing the yield of a plant may include improving the quality of the plant and / or its products. Improving plant quality may include, but is not limited to, improving a particular plant characteristic, such as increasing the content and / or composition of a particular component by a measurable or significant amount over the same factor in a plant produced under the same conditions, but not the composition of the present invention. Improved quality may be characterized by, among others: increased nutritional content; 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; high storage capacity; high processability of harvested products; or any combination thereof.

[0129] Increasing plant yield may include improving the tolerance or resistance of plants to biotic and / or abiotic stress factors. Biotic and abiotic stress can have detrimental effects on plants, especially over time. Biotic stress is caused by organisms, while abiotic stress is caused by, for example, extreme environments. In one embodiment, applying the compounds and / or compositions described herein to plants according to the method of the present invention enhances tolerance or resistance to biotic and / or abiotic stress factors, which means that: (1) the specific negative factor caused by the biotic and / or abiotic stress factor is reduced in a measurable or significant amount compared to a plant exposed to the same conditions, but not treated with the compounds and / or compositions described herein; (2) the negative factor is not reduced by the direct action of the composition against the stress factor (e.g., by its fungicidal or insecticidal action that directly destroys microorganisms or pests, but rather by stimulating the plant's own defense response against the stress factor).

[0130] Negative factors caused by biotic stresses such as pathogens and pests are widespread and range from leaf punctation to the complete destruction of the plant. Biotic stresses can be caused by pests (e.g., insects, arachnids, and nematodes), competing plants (e.g., weeds), microorganisms (e.g., plant pathogenic fungi and / or bacteria), and / or viruses.

[0131] Negative factors caused by abiotic stress are also well known and can often be observed as reduced vigor (as described above) or by any of the following symptoms: spotted leaves, "burned" leaves, reduced growth, reduced number of flowers, reduced biomass, reduced crop yield, reduced nutritional value of the crop, and delayed maturation of the crop (to name just a few). Abiotic stress can be caused by extreme temperature changes such as heat or cold (heat stress / cold stress), strong temperature fluctuations, specific seasonal temperatures, drought (drying stress), extreme humidity, high salinity (salt stress), radiation (e.g., due to increased UV radiation due to a depletion of the ozone layer), elevated ozone levels (ozone stress), organic pollution (e.g., due to toxic amounts of pesticides), inorganic pollution (e.g., due to heavy metal pollutants), and any combination thereof.

[0132] Biotic and / or abiotic stress factors reduce the quantity and quality of the stressed plant, its crops and fruits. As far as quality is concerned, reproductive development can be affected with consequences for crops, important for fruits or seeds. Protein synthesis, accumulation and storage are mainly affected by temperature, growth is slowed down by almost all types of stress, and both structural and storage polysaccharide synthesis is reduced or modified. These effects result in a reduction in biomass (yield) and changes in the nutritional value of the plant products.

[0133] The above-mentioned specific indicators of plant health may be interdependent and may result from each other. For example, increased resistance to biotic and / or abiotic stress may lead to improved plant vigor, e.g., better and larger crops, which may lead to increased yields. Conversely, a more developed root system may lead to increased resistance to biotic and / or abiotic stress.

[0134] Application of the compounds and / or compositions described herein to the plant and / or culture area provides the following synergistic effects to the plant: improved plant health, increased plant yield, increased plant biomass, increased plant oil content, improved plant vigor, increased plant height, increased plant hairiness, enhanced plant root growth, increased plant photosynthetic activity, improved plant quality, improved plant nutritional composition, improved plant protein composition, improved plant carotenoid composition, increased plant resistance to biotic stress, increased plant resistance to fungi, increased plant resistance to nematodes, increased plant resistance to bacteria, increased plant resistance to abiotic stress, increased plant resistance to drought stress, increased plant resistance to cold stress, increased plant resistance to heat stress, increased plant resistance to salt stress, increased plant resistance to ozone stress, and / or any combination thereof.

[0135] One of the most important factors for increasing resistance to biotic and abiotic stresses is the stimulation of the plant's natural defense response, which occurs by applying the compounds and / or compositions described herein according to the methods described herein.

[0136] The plant, including the roots, flowers, leaves, or stems, can be contacted with the disclosed compounds or compositions in any known technique for applying botanicals.

[0137] Exemplary application techniques include, but are not limited to, spraying, spraying, dusting, spreading, watering, dripping, soaking, irrigation, injection, hydroponics, or direct application to water (underwater). In some embodiments, suitable compositions may include compositions for HV, LV, and ULV spraying, as well as ULV cold fog and hot fog formulations. Application methods may vary depending on the intended purpose. The composition may be applied to plants in the field or greenhouse. In some aspects, the composition may be applied to parts of the plant, for example, to tubers before planting. In some embodiments, the compound or composition may be applied to the plant surface or plant plasma membrane as a foliar spray.

[0138] The composition may be contacted with any part of the plant, for example, the roots or leaves of the plant. In some embodiments, the composition may be contacted with the roots by spraying into the soil, mechanically incorporating, mixing with fertilizer, soil amendment, premix, etc.

[0139] In some embodiments, the composition can be contacted with plant seeds. The seed treatment containing the compound or composition can be applied using any commercially available seed treatment machine, or can be applied using any acceptable non-commercial method(s), such as a syringe or any other seed treatment device. The typical seed treatment coating procedure using the compound or composition can be carried out using Wintersteiger HEGE 11 (Wintersteiger AG, Austria, Germany), and can be applied to the seeds of major crops, namely corn, soybean, wheat, rice and various vegetables.

[0140] Seeds can be coated using a variety of methods, including, but not limited to, injecting or pumping an aqueous solution containing the compound or composition onto or through the seed, misting or dusting a layer of seeds, with or without a conveyor system. Suitable mixing devices include tumblers, mixing tanks or drums, or other fluid application devices that contain tanks or drums used to contain the seeds during coating.

[0141] The compositions described herein may be contacted with the plant intermittently. In some aspects, the plant may be contacted with the composition two or more times. For example, the plant may be contacted with the composition 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the plant may be contacted with the composition 2 to about 5 times. In some embodiments, the plant may be contacted with the composition once. In some aspects, the plant may be contacted with the composition once every 5 to 21 days. For example, the plant may 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, the plant may be contacted with the composition once a week. In some aspects, the plant may be contacted with the composition 1 to 5 times a 5 to 21 day period. For example, the plant may be contacted about 1 to about 5 times a week.

[0142] In some aspects, the compositions described herein may be applied before the stress factor(s) occur.

[0143] The term "plant" as used herein includes whole plants and parts thereof, including, but not limited to, vegetative organs / structures of shoots (e.g., leaves, stems and tubers), roots, flowers and floral organs / structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seeds (including embryos, endosperm, and seed coats) and fruits (mature ovaries), plant tissues (e.g., vascular tissue, ground tissue, etc.) and cells (e.g., guard cells, egg cells, etc.), and their progeny. As used herein, "plant cell" refers to any plant cell and may include cells on the plant surface or within the plant cell membrane, such as epidermal cells, trichome cells, xylem cells, phloem cells, phloem elements, or trichome cells, sieve tube elements, or companion cells.

[0144] Plant classifications that may be used in the methods described herein include higher and lower plant classes, including angiosperms (monocotyledons and dicotyledons), gymnosperms, ferns, equinophytes, psilophytes, lycophytes, rice plants, and multicellular algae. For example, plants for use in the methods described herein include any vascular plant, e.g., a monocotyledonous or dicotyledonous plant, or a gymnosperm, including, but not limited to, alfalfa, apple, Arabidopsis, banana, barley, canola, castor, chrysanthemum, clover, cocoa, coffee, cotton, cottonseed, corn, cramberry, cucumber, Dendrobium, Dioscorea, eucalyptus, fescue, flax, gladiolus, lily, linseed, millet, muskmelon, mustard, oats, oil palm, rapeseed, papaya, peanut, pineapple, ornamental plants, Phaseolus, potato, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, sugar beet, sugarcane, 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 trees and nuts such as apple, pear, peach, orange, grapefruit, lemon, lime, almond, pecan, walnut, hazel; vines such as grapes, kiwi, hops; shrub fruits and berries such as raspberries, blackberries, gooseberries; forest trees such as ash, pine, fir, maple, oak, chestnut, with alfalfa, canola, castor, corn, cotton, crambe, flax, linseed, mustard, oil palm, rapeseed, peanut, potato, rice, safflower, sesame, soybean, sugar beet, sunflower, tobacco, tomato, and wheat being popular and preferred. In some embodiments, plants for use in the methods described herein include any crop, such as forage crops, oilseed crops, grain crops, fruit crops, vegetable crops, fiber crops, spice crops, nut crops, turf crops, sugar crops, beverage crops, and forest crops.

[0145] Plants can be classified into agricultural plants, silvicultural plants, ornamental plants, and horticultural plants based on human use and / or consumption. Furthermore, "plants" include natural or wild-type plants, and genetically modified plants.

[0146] "Agricultural" plants are plants that are harvested or cultivated, in part or in whole, on a commercial scale, or that serve as a significant source of feed, food, fiber (e.g., cotton and flax), combustibles (e.g., wood, bioethanol, biodiesel, and biomass), or other compounds. Agricultural plants also include vegetables. Thus, agricultural plants include cereals (e.g., wheat, rye, barley, triticale, oats, sorghum, and rice); beets (e.g., sugar beet or fodder beet), legumes (e.g., beans, lentils, peas, alfalfa, and soybeans); oleaginous plants (e.g., rapeseed, rapeseed, canola, juncea, linseed, mustard, olive, sunflower, cocoa bean, castor bean, oil palm, groundnut, and soybean); cucurbits (e.g., pumpkin, cucumber, and melon); fiber plants (e.g., cotton, flax, hemp, and jute); vegetables (e.g., cucumber, spinach, lettuce, asparagus, cabbage, carrot, radish, turnip, celery). , chicory, endive, Brussels sprouts, parsley, cauliflower, brocoli, garlic, eggplant, pepper, pumpkin, onion, tomato, potato, sweet potato, cucurbits, and paprika; lauraceae plants (e.g., avocado, cinnamon, and camphor); energy and raw materials 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 grape vines); hops; stone fruit; apples; blueberries; strawberries; pears; citrus fruits; raspberries; pineapple; sugar cane; turf; rubber plants, and hemp.

[0147] "Horticultural plants" are plants commonly used in horticulture, including, but not limited to, ornamental plants, vegetables, and fruits. "Ornamental" plants are plants commonly used in horticulture, such as parks, gardens, and balconies and terraces. Non-limiting examples of ornamental plants include grass, geraniums, pelargonias, petunias, begonias, and fuchsias. Non-limiting examples of vegetables are as described above. Non-limiting examples of fruits include apples, pears, cherries, strawberries, citrus fruits, peaches, apricots, and blueberries.

[0148] "Silvicultural" plants are understood to be trees, more specifically trees used in reforestation or industrial plantations. Industrial plantations generally serve the purpose of commercial production of forest products, such as timber, pulp, paper, rubber trees, Christmas trees, or young trees for horticultural purposes. Non-limiting examples of silvicultural plants are conifers (e.g. pines), especially firs and spruces of the Pinus species; eucalyptus; tropical trees (e.g. teak, rubber tree, oil palm); willows (salix), especially Salix species; poplars (poplars), especially Populus species; beeches, especially Fagus species; birch; oil palm; cherry, walnut, and oak.

[0149] As mentioned above, the term "plant" also includes plants that have been modified from wild-type forms. Such modifications can be caused through breeding, mutagenesis, or genetic modification (including transgenic and non-transgenic plants). Genetically modified plants include plants whose genetic material has been modified by the use of recombinant DNA techniques. Such modifications typically include modifications that are not readily obtainable by heterologous breeding, mutation, or natural recombination under natural circumstances. Typically, one or more genes are incorporated into the genetic material of a genetically modified plant to improve a particular characteristic of the plant. Examples of genetically modified plants include, but are not limited to, crops that are resistant to the action of herbicides, fungicides, or insecticides through breeding, including genetic engineering techniques, or plants with modified characteristics compared to existing plants, which can be generated, for example, by conventional breeding methods and / or the generation of mutants, or by recombinant procedures. Examples of genetically modified plants include those that, using recombinant DNA techniques, are able to synthesize one or more proteins to increase the resistance or tolerance of these plants to bacterial, viral, or fungal pathogens; productivity (e.g., biomass production, grain yield, starch content, oil content, and / or protein content), resistance to drought, salinity, or other growth-limiting environmental factors, or resistance of these plants to pests and fungal, bacterial, or viral pathogens; and / or the inclusion of modified or new amounts of ingredients, in particular improved feedstock production, and / or improved human or animal nutrition, for example, increased production of potatoes that produce increased amounts of amylopectin (e.g., oil crops that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids (e.g., Nexera® canola, DOW Agro Sciences, Canada), AmFlora® potato, BASF SE, Germany).

[0150] Methods for producing such genetically modified plants are generally known to those skilled in the art and are described, for example, in the references mentioned above.

[0151] Compositions, Formulations and Methods of Administration Also disclosed herein are compositions that include the compounds disclosed herein.

[0152] Agronomic formulations of active substances are well known. Non-limiting examples include solids, semi-solids, liquids, solutions, suspensions, emulsions, gels, oil dispersions, capsules (such as microencapsulated active ingredients), dusts, pastes, granules, and the like. The particular formulation selected may vary depending on the particular intended mode of administration. In either case, it is usually advantageous to ensure a fine and uniform distribution of the active ingredient(s) in the liquid or solid carrier. The compositions described herein may be in any suitable form based on their intended use. In some aspects, the composition may be in the form of an aqueous solution. In some aspects, the composition may be a solution comprising an organic solvent, such as an alcohol. In some aspects, the composition may be a solution comprising a mixture of an organic solvent and an inorganic solvent.

[0153] Preferably, the compositions described herein are formulated in a manner suitable for large- or small-scale agricultural and horticultural applications.

[0154] In particular, the dosage level of the composition selected will depend on various factors well known in the agricultural field, such as, for example, route of administration, time of administration, duration of treatment, other drugs and / or substances used in combination with the specific compound used, and the disease state and health state of the plant being treated.However, the compositions described herein provide plant stimulation even at low doses.Those skilled in the art with ordinary skill in the art can easily determine and formulate the effective amount of the composition required.

[0155] Formulation methods are described, 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 91 / 13546 to EI Du Pont De Nemours and Co.; U.S. Pat. No. 4,172,714 to Albert, U.S. Pat. No. 4,144,050 to Frensch et al.; U.S. Pat. No. 3,920,442 to Albert, U.S. Pat. No. 5,180,587 to Moore; U.S. Pat. No. 5,232,701 to Ogawa et al.; Hoy et al. No. 5,208,030 to Hance et al., British Patent No. 2,095,558; 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 Ed., 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.

[0156] The composition may comprise an agriculturally effective amount of the compound described herein in combination with an agriculturally acceptable carrier, as described above, and may also comprise other adjuvants.The composition may be formulated in a manner that is common to agrochemical formulations (either together or separately).For example, the composition(s) may comprise adjuvants that are common to agrochemical formulations.The specific adjuvants used may depend on the specific application form and active substance, respectively. Non-limiting examples of suitable auxiliary agents include carriers, adjuvants, diluents, thickeners, buffers, preservatives, surfactants, wetting agents, coating agents, monosaccharides, polysaccharides, abrasives, repellents, insecticides, herbicides, nematicides, bactericides, fungicides, acaricides, fertilizers, biostimulants, colorants, humectants, antifreeze agents, antifoam agents, compatibilizers, sequestering agents, neutralizing agents and buffers, rust inhibitors, osmoprotectants, odorants, antibiotics, spreading agents, dispersants, freezing point depressants, antimicrobial agents, crop oils, safeners, adhesives, protective colloids, emulsifiers, tackifiers, amino acids, biological pesticides, or combinations thereof.

[0157] In some embodiments, the compositions described herein may include a pesticide, including an insecticide, herbicide, fungicide, bactericide, nematicide, acaricide, or any combination thereof. The pesticide may be applied to the plant simultaneously or sequentially. In some embodiments, the pesticide is applied to the plant after the compounds and / or compositions described herein are applied.

[0158] In some aspects, a concentrate suitable for dilution of the composition may be prepared using the composition in addition to water, a wetting agent, a tackifier, a dispersing agent, or an emulsifier.

[0159] Agriculturally acceptable carriers may include organic or inorganic carriers. Exemplary carriers include water, organic solvents, inorganic solvents, hydrocarbons such as petroleum fractions or mineral oils, aromatic solvents, vegetable oils such as paraffin oil, 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, and tung oil, esters of the above vegetable oils, monohydric, dihydric, and trihydric alcohols, or other lower polyalcohols (containing 4 to 6 hydroxyl groups), such as 2-ethylhexyl stearate, n-butyl oleate, isopropyl myristate, propylene glycol dioleate, dioctyl succinate, dibutyl adipate, and dioctyl phthalate esters. Examples of suitable organic solvents include, but are not limited to, esters of ter, mono, di, and polycarboxylic acids, toluene, xylene, petroleum 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, methyl alcohol, ethyl alcohol, isopropyl alcohol, amyl alcohol, ethylene glycol, propylene glycol, glycerin, N-methyl-2-pyrrolidinone, N,N-dimethylalkylamides, dimethyl sulfoxide, liquid fertilizers, and mixtures thereof. Other exemplary carriers include silica, silica gel, silicates, talc, kaolin, limestone, lime, chalk, agglomerate clay, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, pyrophyllite clay, attapulgas clay, diatomaceous earth, calcium carbonate, bentonite clay, fuller's earth, cottonseed kernels, wheat flour, soy flour, light flour, wood flour, walnut shell flour, lignin, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, grain flour, bark flour, wood and nut shell powders, cellulose powder, and mixtures thereof.The agriculturally acceptable carrier may be present in an amount of 99.9% by weight or less, 99% by weight or less, 98% by weight or less, 97% by weight or less, 95% by weight or less, 90% by weight or less, 85% by weight or less, 80% by weight or less, 75% by weight or less, 70% by weight or less, 65% by weight or less, 60% by weight or less, 55% by weight or less, 50% by weight or less, 45% by weight or less, or 40% by weight or less, based on the weight of the composition.

[0160] When the composition comprises an amino acid, the amino acid can be provided separately from the amino acid that comprises the polypeptide. For example, isolated amino acids can be used. Suitable amino acids include any natural or unnatural amino acid. For example, the composition can comprise cysteine.

[0161] Unless otherwise specified, each agriculturally acceptable adjuvant may be present at 0.1-60%, 0.5-50%, or 10-30% by weight of the total weight of the composition.

[0162] If the composition includes a preservative, the preservative may include dichlorophene and benzyl alcohol hemiformal (PROXEL from ICI or ACTICIDE RS from Thor Chemie and KATHON MK from Dow Chemical) and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones (ACTICIDE MBS from Thor Chemie). By way of further example, suitable preservatives include MIT (2-methyl-4-isothiazolin-3-one), BIT (PROXEL from Avecia, Inc.), GXL (available as a solution in sodium hydroxide and dipropylene glycol), 1,2-benzylisothiazolin-3-one), 5-chloro-2-(4-chlorobenzyl)-3(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.

[0163] When the composition includes a buffer, the buffer can include potassium, phosphoric acid, phosphate, citric acid, citrate, sulfate, MOPS, or HEPES. The buffer can stabilize the polypeptide in the composition.

[0164] If the composition includes a wetting agent, the wetting agent may include organosilicones, polyoxyethoxylates, polysorbates, polyethylene glycols and their derivatives, ethoxylates, crop oils, and polysaccharides.

[0165] If 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, polyhydric alcohols, alkyl phosphates, alcohol ethoxylates, alkylphenol ethoxylates, alkylphenol ethoxylates, alkoxylated polyols, alkyl polyethoxy ethers, alkyl polyoxyethylene glycerols, ethoxylated derivatives and soybean oil derivatives, organosilicon surfactants, or any combination thereof. Surfactants may be included in a variety of compositions, including those for foliar use.

[0166] When the composition includes a coating, the coating may include a tackifier, a polymer, a filler, or an extender.

[0167] Tackifiers 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, as well as natural phospholipids, such as cephalin and lecithin, and synthetic phospholipids. Tackifiers include those that are composed of adhesive polymers, which may be natural or synthetic, preferably without phytotoxicity to the seed being coated. Additional tackifiers that may be included alone or in combination include, for example, polyesters, polyetheresters, polyanhydrides, polyesterurethanes, polyesteramides; polyvinyl acetates; polyvinyl acetate copolymers; polyvinyl alcohol and tyloses; polyvinyl alcohol copolymers; polyvinylpyrrolidone; starches, modified starches and starch derivatives, dextrins, maltodextrins, alginic acid, chitosan, and cellulose, cellulose esters, cellulose ethers, and polysaccharides, including ethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, and carboxymethylcellulose. Examples of suitable tackifiers include cellulose ether esters, including fats, oils, proteins, including casein, gelatin, and zein, gum arabic, shellac, vinylidene chloride and vinylidene chloride copolymers, lignosulfonates, especially calcium lignosulfonate, polyacrylates, polymethacrylic acid and acrylic copolymers, polyvinyl acrylate, polyethylene oxide, polybutene, polyisobutene, polystyrene, polybutadiene, polyethyleneamine, polyethyleneamide, acrylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomer, and polychloroprene, or any combination thereof. The tackifiers may be used in a variety of compositions, including those for seed treatment.

[0168] If the composition includes an abrasive, the abrasive can include talc, graphite, or a combination of both.

[0169] Moisturizing agents are hygroscopic substances that help retain moisture.When the composition comprises a humectant, the humectant can include glycerol, glycerin, glycerol derivatives (e.g., glycerol monostearate, glycerol triacetate, triacetin, propylene glycol, hexylene glycol, or butylene glycol), triethylene glycol, tripolypropylene glycol, glyceryl triacetate, sucrose, tagatose, sugar alcohol or sugar polyol (e.g., glycerol, sorbitol, xylitol, mannitol, or mantitol), polymeric polyol (e.g., polydextrose, collagen, aloe or aloe vera gel), or alpha hydroxy acid (e.g., lactic acid, honey, molasses, quillaja, sodium hexametaphosphate, lithium chloride, or urea).Synthetic humectants can also include butylene glycol, and tremella extract.

[0170] When the compounds described herein are formulated or applied in combination with a commercial fungicide, the composition can provide an additional layer of protection to enhance the prevention or spread of disease in plants.

[0171] A variety of colorants can be used including organic chromophores classified as nitroso, nitro, azo including monoazo, bisazo, and polyazo, diphenylmethane, triarylmethane, xanthene, methane, acridine, thiazole, thiazine, indamine, indophenol, azine, oxazine, anthraquinone, phthalocyanine, or any combination thereof.

[0172] A biopesticide is broadly defined as a microorganism that can be used in place of synthetic pesticides or fertilizers. When the composition includes a biopesticide, the biopesticide can include Bacillus thuringiensis, Bacillus megaterium, Bacillus mycoides isolate J, Bacillus methylotrophicus, Bacillus vallismortis, Chromobacterium subtsugae, Delftia acidovorans, Streptomyces lydicus, Streptomyces colombiensis, Streptomyces galbus K61, Penicillium bilaii. It can include lipopeptide-producing Bacillus subtilis strains, lipopeptide-producing Bacillus amyloliquefaciens strains, Bacillus firmus strains, or Bacillus pumilus strains.

[0173] When the composition includes a fertilizer, the fertilizer may be selected from the group consisting of 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, ammonium calcium nitrate, calcium sulfate, calcined magnesia, calcitic limestone, calcium oxide, calcium nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate. , potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrate, dolomite, magnesia, urea, urea formaldehyde, ammonium urea nitrate, sulfur coated urea, polymer coated urea, isobutylidenediurea, K2SO4-Mg2SO4, kainite, sylvinite, kieselite, 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, green sand, cottonseed meal, feather meal, crab shell meal, fish fertilizer, humic acid, or any combination thereof.

[0174] The fertilizer may include a liquid fertilizer or a dry fertilizer. The composition may include a micronutrient fertilizer material comprising boric acid, a borate salt, a boron frit, copper sulfate, copper frit, copper chelate, sodium tetraborate decahydrate, ferrous sulfate, ferrous oxide, ammonium ferrous sulfate, 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.

[0175] If the composition includes a biostimulant, the biostimulant may include a seaweed extract, an elicitor, a polysaccharide, a monosaccharide, a protein extract, a soybean extract, humic acid, a plant hormone, a plant growth regulator, or any combination thereof.

[0176] Examples of thickeners (i.e. compounds that impart modified 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).

[0177] Examples of suitable antifreeze agents are ethylene glycol, propylene glycol, urea, and glycerin.

[0178] Examples of antifoaming agents are silicone emulsions (e.g., Silikon® SRE, Wacker, Germany, and Rhodorsil®, Rhodia, France), long chain alcohols, fatty acids, salts of fatty acids, fluoroorganic compounds, and mixtures thereof.

[0179] Antifungal agents include aldimorph, ampropylphos, ampropylphos potassium, andoprim, anilazine, azaconazole, azoxystrobin, benalaxyl, benodanil, benomyl, benzamacryl, benzamacryl-isobutyl, benzovindiflupyr, bialaphos, binapacryl, biphenyl, bitertanol, blasticidin-S, boscalid, bromuconazole, bupirimate, buthiobate, calcium polysulfide, capsimycin, captafol, captan, carbendazim, carvone, chinomethionate, clobenzylazon, and chlorof. Enazol, chloroneb, chloropicrin, chlorothalonil, clozolinate, clozylacon, khufraneb, cymoxanil, cyproconazole, cyprodinil, cyproflam, debacarb, dichlorophen, diclobutrazol, dichlofluanid, diclomedine, dicloran, diethofencarb, dimetrimol, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinocap, diphenylamine, dipyrithione, ditalimphos, dithianon, dodemorph, dodine, drazoxolone, edifenphos, epoxiconazole, ethoxyconazole, Taconazole, Ethirimol, Etridiazole, Famoxadone, Fenapanil, Fenarimol, Fenbuconazole, Fenfuram, Fenitropan, Fenpiclonil, Fenpropidin, Fenpropimorph, Triphenyltin Acetate, Fentin Hydroxide, Ferbam, Ferimzone, Fluazinam, Fludioxonil, Flumetober, Fluoromid, Fluoxastrobin, Fluquinconazole, Flurprimidol, Flusilazole, Flusulfamide, Flutolanil, Flutriafol, Folpet, Fosetylaluminum, Fosetylsodium um, phthalide, fuberidazole, furalaxyl, furametpyr, flucarbonyl, fluconazole, fluconazole-cis, flumecyclox, guazatine, hexachlorobenzene, hexaconazole, hymexazole, imazalil, imibenconazole, iminoctadine, iminoctadine albesilate, iminoctadine triacetate, iodocarb, iprobenfos (IBP), iprodione, irumamycin, isoprothiolane, isovalerion, kasugamycin, kresoxim methyl, copper hydroxide, copper naphthenate, copper oxychloride, copper sulfate,Copper oxide, copper oxine and other copper preparations, as well as Bordeaux mixture, mancopper, mancozeb, maneb, meferimzone, mepanipyrim, mepronil, metconazole, metalaxyl, methasulfocarb, metofloxam, metiram, metomeculam, methsulfovax, mildiomycin, myclobutanil, myclozolin, myclozolin, nitrosal-isopropyl, nuarimol, ofurace, oxadixyl, oxamocarb, oxolinic acid, oxycarboxim, oxyfenthiin, paclobutrazol, pefurazoate, penconazole, peneconazole, Ncicloron, phosdifen, phosdifen, picoxystrobin, pimaricin, piperalin, polyoxin, polyoxorim, probenazole, prochloraz, procymidone, propamocarb, propanocin sodium, propiconazole, propineb, prothiocyanazole, pyrazophos, pyrifenox, pyrimethanil, piroquilon, piroxyflur, quinconazole, quintozene (PCNB), strobilurin, sulfur and sulfur preparations, tebuconazole, tecloftalam, tecnazene, tetcyclasis, tetraconazole, thiabenda azole, thiofen, thifluzamide, thiophanate-methyl, tioximide, tolclofos-methyl, tioximide, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazbutyl, triazole, triazoxide, triclamide, tricyclazole, triclopyr, tridemorph, trifloxystrobin, triflumizole, triforine, uniconazole, validamycin A, vinclozolin, viniconazole, zaliramide, zineb, as well as Dagger G, OK-8705, OK-8801, α-( 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-dioxan-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-naphthalene) phthalenyl)-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]-ethenyl]-1H -imidazole, 1-methyl-5-nonyl-2-(phenylmethyl)-3-pyrrolidinol, 2',6'-dibromo-2-methyl-4'-trifluoromethoxy-4'-trifluoro-methyl-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-glycopyranosyl)-aD-glucopyranosyl]-amino]-4-methoxy-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile, 2-aminobutane, 2-bromo-2-(bromomethyl)-pentanedinitrile,2-Chloro-N-(2,3-dihydro-1,1,3-trimethyl-1H-inden-4-yl)-3-pyridinecarboxamide, 2-Chloro-N-(2,6-dimethylphenyl)-N-(isothiocyanatomethyl)-acetamide, 2-phenylphenol (OPP), 3,4-dichloro-1-[4-(difluoromethoxy)-phenyl]-pyrrole-2,5-dione, 3,5-dichloro-N-[cyano[(1-methyl-2-propynyl)-oxy]-methyl]-benzamide, 3-(1,1-dimethylpropyl-1-oxo-1H-inden- 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-methyl-tetrazolo[1,5-a]quinazolin-5(4H)-one, 8-(1,1-dimethylethyl)-N-ethyl-N-propyl-1,4-dioxaspiro[4,5]decane-2-methanamine, 8-hydroxyquinoline sulfate, 9H-xanthene-2-[(phenylamino)-carbonyl]-9- Carboxylic acid hydrazide, bis-(1-methylethyl)-3-methyl-4-[(3-methylbenzoyl)-oxy]-2,5-thiophenedicarboxylate, 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, 1H-tetramethyl-2-phenylpropanediol ... -(2,3-dihydro-2,2-dimethyl-inden-1-yl)-1H-imidazole-5-carboxylate methyl, N-(2,6-dimethylphenyl)-N-(5-isoxazolylcarbonyl)-DL-alaninate methyl, N-(chloroacetyl)-N-(2,6-dimethylphenyl)-DL-alaninate methyl, 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-nitro-benzenesulfonamide, 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-pyridinyl)-cyclopropanecarboxamide, N-[2,2,2-trichloro-1-[(chloroacetyl)-amino]-ethyl]-benzamide, N-[3-chloro-4,5-bis(2-propynyloxy)-phenyl]-N'-methoxy-methanamide imide, N-formyl-N-hydroxy-DL-alanine-sodium salt, O,O-diethyl[2-(dipropylamino)-2-oxoethyl]-ethyl phosphoroamidothioate, O-methyl S-phenylphenylpropyl phosphoroamidothioate, S-methyl 1,2,3-benzothiadiazole-7-carbothioate, and spiro[2H]-1-benzopyran-2,1'(3'H)-isobenzofuran]-3'-one, N-trichloromethyl)thio-4-cyclohexane-1,2-dicarboximide, tetramethylthioperoxydicarbonic diamide, methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate, 4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1-H-pyrrole-3-carbonitrile, or any combination thereof.

[0180] 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.

[0181] The strobilurin fungicide may include a non-naturally occurring strobilurin fungicide such as azoxystrobin, trifloxystrobin, kresoxim-methyl, fluoxastrobin, or any combination thereof. For example, the strobilurin fungicide may include trifloxystrobin, fluoxastrobin, or picoxystrobin. Strobilurin fungicides are used to control a variety of fungal diseases including aquatic mold, downy mildew, powdery mildew, spot and dieback mold, fruit rot, and rust. They are useful for treating a variety of crops including cereals, field crops, fruits, nuts, vegetables, turfgrass, and ornamentals.

[0182] Triazole fungicides may include prothioconazole, imidazole, imidazyl, prochloraz, propiconazole, triflumizole, diniconazole, flusilazole, penconazole, hexaconazole, cyproconazole, myclobutanil, tebuconazole, difenoconazole, tetraconazole, fenbuconazole, epoxiconazole, metconazole, fluquinconazole, triticonazole, or any combination thereof.

[0183] Additionally, fungicides may include azoxystrobin, carboxin, difenoconazole, fludioxonil, fluxapyroxad, ipconazole, mefenoxam, pyraclostrobin, silthiofam, sedaxane, thiram, triticonazole, or any combination thereof.

[0184] Herbicides include 2,4-D, 2,4-DB, acetochlor, acifluorofen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulfuron methyl bensulide, bentazone, bispyribac sodium, bromacil, bromoxynil, butyrate, carfentrazone, chlorimuron, 2-chlorophenoxyacetic acid, chlorsulfuron, chlorimuron ethyl, cletozine, clomazone, clopyralid, chloransulam, CMPP-P-DMA, cycloate, DCPA, desmedipham, dicamba, dichlobenil, diclofop, 2,4-dichlorophenol, dichlorophenoxyacetic acid, dichloropropa, dichlorprop, dichlorprop-P, diclosulam, diflufenzopyr, dimethenamid, 2,Dimethylamine salt of 4-dichlorophenoxyacetic acid, diquat, diuron, DSMA, endothal, EPTC, ethalfluralin, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, floroxypyr 1-methylputilester, fomesafen, fomesafen sodium salt, formsulfuron, glufosin 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, mecoprop, mecoprop-P, mesotrione, metolachlor-s, mecoprop-p The active ingredients may include tribuzin, metsulfuron, metsulfuron-methyl, molinate, MSMA, napropamide, naphthalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propanil, prosulfuron, pyrazone, pyrithiobac, pyroxasulfone, quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thifensulfuron-methyl, thiobencarb, tralkoxydim, triallate, triasulfuron, tribenuron, tribenuron-methyl, triclopyr, trifluralin, triflusulfuron, or any combination thereof. ,

[0185] Where the composition comprises a nematicide, the nematicide may be selected from the group consisting of antibiotic nematicides such as Bacillus firmus, fluopyram, and abamectin; carbamate nematicides such as acetoprole, Bacillus chitonosporus, chlorpicrin, benclothiaz, benomyl, Burholderia cepacia, carbofuran, carbosulfan, and creosocard; nematicides such as dazomet, DBCP, DCIP, alanycarb, aldicarb, aldoxycarb, oxamyl, diamidaphos, fenamiphos, hosthietane, phosphamidon, cadusafos, chlorpyrifos, diclofenthion, dimethoate, ethoprophos, fensulfothion, hosthiazate, harpin, heterophos, imicyaphos, isamidophos, isazophos, methomyl, Myrothecium verrucaria, Paecilomyces lilacinus, Pasteuria For example, the nematicide may include Bacillus firmus strain i-2580, Pasteuria nishizawae (including its spores), phorate, phosphocarb, terbufos, thionazine, triazophos, thioxazaphen, dazomet, 1,2-dichloropropane, 1,3-dichloropropene, furfural, iodomethane, metham, methyl bromide, methyl isothiocyanate, xylenol, or any combination thereof.

[0186] If the composition includes a bactericide, the bactericide may include streptomycin, penicillin, tetracycline, oxytetracycline, kasugamycin, ampicillin, oxolinic acid, chlorotetracycline, copper oxide, or any combination thereof. For example, the bactericide may include oxytetracycline.

[0187] If the composition includes an insecticide, the insecticide may include clothianidin, imidacloprid, an organophosphate ester, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, a boric acid, a borate, a fluoride, a sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically based insecticide, or any combination thereof. For example, the insecticide may include clothianidin or imidacloprid.

[0188] If the composition includes an insecticide, the insecticide may include an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, a sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically based insecticide, or any combination thereof.

[0189] Also disclosed are kits comprising the compounds disclosed herein in one or more containers. The disclosed kits may optionally include an agriculturally acceptable carrier and / or diluent. In one embodiment, the kit includes one or more other ingredients, supplements, or adjuvants described herein. In one embodiment, the kit includes instructions or packaging materials that describe how to administer the compounds or compositions of the kit. The containers of the kit can be of any suitable material, e.g., glass, plastic, metal, and any suitable size, shape, or configuration. In one embodiment, the compounds and / or agents disclosed herein are provided in the kit as solids, such as tablets, pills, or powder forms. In another embodiment, the compounds and / or agents disclosed herein are provided in the kit as liquids or solutions.

[0190] Numerous embodiments of the invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES

[0191] The following examples are presented so as to provide those of skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed in this disclosure are made and evaluated, and are intended to be merely exemplary of the invention and are not intended to limit the scope of what the inventors regard as the invention. However, those of skill in the art should, in light of this disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0192] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0193] Peptides and proteins are generally impermeable to plant cell membranes. An engineered human membrane translocation domain, MTD4, can efficiently enter the cytosol of plant cells. Genetic fusion of MTD4 to the bacterial harpin HrpZ results in efficient delivery of HrpZ to tobacco and tomato plants, causing cell death and enhanced resistance to pathogens.

[0194] To investigate whether MTD4 can penetrate plant tissues and cells, we expressed and purified MTD4, and labeled the parent protein (FN3) from Escherichia coli with tetramethylrhodamine (TMR) (Liss, V., et al., Sci Rep 5 (2016)). TMR and FN3 TMR was sprayed onto N. benthamiana leaves that had been infiltrated with Agrobacterium expressing green fluorescent protein (GFP). Confocal microscopy imaging 2 hours after spraying revealed that the MTD4 TMR Intense red fluorescence was observed inside the cells of N. benthamiana leaves treated with FN3 (Fig. 3B, right panel), whereas FN3 TMRHowever, this was not observed in leaves treated with MTD4 (Fig. 3B, left panel). As expected, GFP was expressed and localized in the cytosol of the cells. TMR Colocalization of green and red fluorescent signals at the cell membrane and inside the cytoplasm of sprayed leaves (FIG. 3B, lower right panel) demonstrates that MTD4 efficiently enters the cytosol of N. benthamiana cells.

[0195] Harpin proteins are produced by Gram-negative bacteria and secreted via a type III secretion system, functioning as elicitors of hypersensitive response (cell death), immune response, and plant growth enhancers (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 crop plants. In fact, harpin-related products have been developed for crop plants for the past 20 years. However, although effective in some applications, these products have not been widely used in crop production because the proteins do not efficiently penetrate plant tissues and therefore have very low bioavailability in foliar applications (Nadendla, SR, et al., Carbohyd Polym 199, 11-19 (2018)).

[0196] To test whether MTD4 could improve the penetration of cargo proteins into plant tissues, HrpZ (He, SY, et al., Cell 73, 1255-1266 (1993)) was genetically fused to the C-terminus of MTD4 and the MTD4-HrpZ fusion protein from E. coli was purified. Treatment of N. tabacum leaves (in foliar application) with 10 μM MTD4-HrpZ (but not HrpZ or MTD4) for 24 h resulted in strong cell death in the leaves (Figure 4A, left panel). In contrast, when N. tabacum leaves were infiltrated with MTD4-HrpZ, HrpZ, or MTD4, strong cell death was observed in either MTD4-HrpZ-treated or HrpZ-treated leaves (Figure 4A, right panel), confirming the ability of HrpZ to induce cell death alone when delivered to the cytosolic space. We also found that spraying Arabidopsis plants with MTD4-HrpZ induced strong cell death in the leaves 24 h after treatment (Figure 5). These results demonstrate that MTD-HrpZ, but not HrpZ, readily enters N. tabacum and Arabidopsis cells and induces cell death through foliar application.

[0197] Effects of MTD4-HrpZ on plant immunity. N. tabacum leaves were sprayed with MTD4-HrpZ and stained with 3,3'-diaminobenzidine (DAB), which detects the levels of reactive oxygen species (ROS) in plant tissues. Treatment with MTD4-HrpZ resulted in significantly more ROS accumulation than those treated with HrpZ (Figure 4B). Furthermore, qRT-PCR analysis showed that the expression of two pathogenesis-related genes, NtHSR203 and NtCHN50, in N. tabacum was significantly upregulated only 3 h after spraying with MTD4-HrpZ compared to those sprayed with HrpZ (Figure 4C). To test whether MTD4-HrpZ could enhance the resistance of N. tabacum to the pathogenic bacterium, P. syringae pv. tomato (Pst) DC3000 hrcC-, N. tabacum plants were sprayed with MTD4-HrpZ, HrpZ, or MTD4 and bacterial titers were measured 3 days post inoculation (dpi). Leaves sprayed with MTD4-HrpZ contained approximately one order of magnitude fewer Pst bacteria than leaves treated with either HrpZ or MTD4 (Figure 4D).

[0198] Botrytis cinerea is one of the most destructive fungal pathogens affecting many plant species, including tomato (Cheung, N., et al., Pathogens 9, 923 (2020)). We tested whether MTD4-HrpZ could protect tomato fruits from B. cinerea. Fresh tomatoes were sprayed with MTD4-HrpZ, HrpZ, or MTD4. After 24 h, the lesion sites of the tomatoes were inoculated with mycelial blocks of B. cinere. Three days after inoculation, tomatoes treated with MTD4-HrpZ showed obviously less growth of B. cinerea than those treated with either HrpZ or MTD4 (Figure 4E). The lesion size of tomatoes treated with MTD4-HrpZ was two-fold smaller than those of the two controls (Figure 4F).

[0199] It was demonstrated that cell-penetrating peptide / protein (CPP) technology can significantly increase the cell entry efficiency of defense activators into plant cells. The cell penetration efficiency of Lys-containing CPPs was higher in plants than in animal cells. This may be due to the difference in lipid composition and membrane surface charge in these two cell types. MTD4 efficiently enters plant cells and significantly increases the cell entry efficiency of HrpZ into plant cells. MTD4 can be genetically fused to any cargo peptide / protein, and the resulting fusion protein can be easily produced on a large scale and cost-effectively through fermentation, so it has high potential to deliver defense activators and biostimulants for foliar applications and seed treatments in crop production.

[0200] method 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 verified by sequencing. [Table 3] [Table 4]

[0201] Plant material and growth conditions. Tobacco and Arabidopsis seeds were sown in soil pots and plants were grown in a growth room at 25° C. under an 8 h / 16 h photoperiod.

[0202] Design, expression, and purification of MTD. 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 a scaffold for binding proteins. J. Mol. Biol. 284 (4): 1141-1151). FN3 is a small (90-100 aa) and highly stable protein that has been widely used to develop monobodies that bind to target proteins with high affinity and specificity (Chandler, PG, et al. (2020) Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain. Cells 9 (3): 610). Previous studies have demonstrated that some loop regions of FN3 are resistant to mutations (Steven, A., et al. (2012) Design of novel FN3 domains with high stability by a consensus sequence approach, Protein Engineering, Design and Selection, 25(3): 107-117). In addition, FN3 is stable in the intracellular environment because it does not have any cysteine ​​or disulfide bonds. FN3 easily folds into its native form without physical or chemical assistance and can be produced in high yields in Escherichia coli. Finally, FN3 is derived from an abundant human extracellular protein and is unlikely to elicit an immune response.

[0203] 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 in the FG loop was replaced with RRRWWW (SEQ ID NO: 104) to obtain MTD1 (Table 5). This, together with the arginine residue already in the FG loop, generates a putative CPP motif (R4W3) without changing the loop size. Similarly, the tetrapeptide AVTV in the BC loop was replaced with WWWRRR (SEQ ID NO: 105) to take advantage of the existing arginine in the loop and form a putative CPP, W3R4 (Table 5). The size of the BC loop of the resulting mutant MTD2 is increased by two residues. To investigate the possibility of grafting a CPP motif to the other end of FN3, the tripeptide NSP in the CD loop was replaced with the CPP motif R4W3 to obtain MTD3. To test the feasibility of grafting the CPP sequence into two different loops, the relatively hydrophobic tripeptide in the BC loop (VTV) was replaced with a hydrophilic motif in the WYW and FG loops, such that RRR generated MTD4 (GDSPAS, SEQ ID NO: 106). Finally, MTD5 was generated by exchanging the WYW and RRR motifs of MTD4. Two mutants, MTD4a and MTD4b, containing only half of the CPP motif in the BC and FG loops, respectively, were also generated to test the relative importance of the RRRR and WYW motifs. The WYW motif is more hydrophilic than WWW and has been previously reported as an "endosomal escape motif" for cell-penetrating antibodies (Kim, J.-S., et al. (2016) Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape. J. Control. Rel. 235: 165-175). The loop insertion mutants were analyzed with the online program Phyre2 to predict their folded structures based on sequence homology. All mutants maintained an overall fold similar to wild-type FN3, with the CPP motifs displayed on the surface and constrained to a "ring" topology (Figure 1).

[0204] 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 total cell entry efficiency of MTD6-10 was assessed by labeling the MTDs at their unique C-terminal cysteines with tetramethylrhodamine-5-maleimide (TMR). HeLa cells were treated with TMR-labeled proteins (5 μM) for 2 h and analyzed by live-cell confocal microscopy. MTD7 TMR and MTD9 TMR MTD4 TMR showed similar uptake to MTD6 TMR , MTD8 TMR and MTD10 TMR showed much less cell entry. Moreover, the isolation yield of MTD6-10 varied from 0.6 to 6.2 mg per L of E. coli cell culture (Table 4). Note that MTD4 and MTD6 only differ slightly in the BC loop sequence ("WYW" vs. "YWW"), but have dramatic differences in isolation yield (9.4 mg / L vs. 0.9 mg / L) as well as cell entry efficiency. Similarly, swapping the CPP motif between the BC and FG loops of MTD4 resulted in a poorly expressed and much less active variant (MTD5 in Table 5). These results demonstrate that proper folding / stability and high cell entry efficiency of MTDs require not only the presence of amphipathic CPP motifs but also their proper presentation on the protein surface.

[0205] A DNA sequence encoding WT FN3 was chemically synthesized and ligated into the prokaryotic expression vector pET-15b. To facilitate protein purification and gene fusion with cargo proteins, a six-histidine 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 the 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 E. coli. Among the mutant proteins, MTD1 failed to produce significant amounts of soluble protein, while WT FN3 and MTD2-5 produced soluble proteins in good yields (Table 5). Figures 2A-2B show the expression and purification of MTD4 as an example. All proteins were purified to near homogeneity by metal affinity chromatography on a Ni-NTA column. [Table 5]

[0206] 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 (Table 6) was confirmed by sequencing the entire coding region of the plasmid DNA. Pilot-scale protein expression was performed to confirm the expression level of the mutant proteins, and all mutants were expressed in 5 mL of E. coli BL21(DE3) bacterial cultures. Induction was performed at 37°C in the presence of 0.25 mM IPTG. Expression levels were checked by comparing pre- and post-induction total cell lysates on SDS gels (Figures 2A-2B). [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0207] The large-scale expression conditions were the same as those used for the small scale, and E. coli cells were centrifuged and stored at -80°C. The cells were lysed using lysis buffer (50 mL of 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 parate was homogenously resuspended in lysis buffer, the cells were sonicated twice (Amp. 70%). The crude cell lysate was centrifuged (12000 g for 20 min) and the soluble cell lysate was collected. Protein purification was performed using fast protein liquid chromatography (FPLC), and the soluble cell lysate was loaded onto a Ni-NTA column (containing 15 mM imidazole). The column was washed extensively with wash buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 5% glycerol, and 50 mM imidazole). Proteins were eluted with wash buffer containing a linear gradient of 50-500 mM imidazole (pH 7.4) over 30 min.

[0208] MTD4 plasmid construction. DNA sequences encoding the 10th FN3 domain and HrpZ were chemically synthesized and ligated into the prokaryotic expression vector pET-15b (MilliporeSigma), which had been linearized using the restriction endonucleases NdeI and XhoI. This cloning procedure also added a flexible linker sequence, (GGS)3, a restriction site (SacI), and a cysteine ​​residue to the C-terminus of FN3. The method for site-directed mutagenesis of FN3 to generate plasmid pET-15b-MTD4 has been described previously (Qi, D.; Scholthof, K.-B. G., 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 products were digested with SacI and XhoI restriction enzymes and ligated into plasmid pET-15b-MTD4 linearized with the same two enzymes. All constructs contain a six histidine tag at the N-terminus for easy purification. The authenticity of the DNA constructs was confirmed by restriction mapping and sequencing of the entire coding sequence.

[0209] HrpZ protein sequence SEQ ID NO: 146 MQSLSLNSSSLQTPAMALVLVRPEAETTGSTSSKALQEVVVKLAEELMRNGQLDDSSPLGKLLAKSMAADGKAGGGIEDVIAALDKLIHEKLGDNFGASADSASGTGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDMPMLNKIAQFMDDNPAQFPKPDSGSWVNE LKEDNFLDGDETAAFRSALDIIGQQLGNQQSDAGSLAGTGGGLGTPSSFSNNSSVMGDPLIDANTGPGDSGNTRGEAGQLIGELIDRGLQSVLAGGGLGTPVNTPQTGTSANGGQSAQDLDQLLGGLLLKGLEATLKDAGQTGTDVQSSAAQIATLLVSTLLQGTRNQAAA HrpZ DNA sequence (SEQ ID NO: 155) based on GenBank accession number: HM358042.1

[0210] Expression and purification of recombinant proteins. E. coli BL21(DE3) cells transformed with the appropriate plasmids were grown in Luria-Bertani (LB) medium supplemented with 75 μg / mL ampicillin at 37 °C. OD 600 When the NA reached 0.6, cells were induced by addition of 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 6 h at 37°C. Cells were pelleted by centrifugation (4000g for 30 min) and resuspended in lysis buffer (50 mL of wash buffer per L of cell culture, 0.2 mg / mL lysozyme, 2 mM β-mercaptoethanol, 2 mM PMSF, 2 tablets of Roche, cOmplete protease inhibitor cocktail). Cells were sonicated briefly and the crude cell lysate was centrifuged (12000g for 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. Protein was eluted with wash buffer containing a linear gradient of 50-500 mM imidazole over 30 min. Purity of the eluted fractions was assessed by SDS-PAGE, and pure protein fractions were combined, concentrated in centrifugal filter units (Millipore), and dialyzed against wash buffer. Protein concentration was determined by Bradford assay. Purified protein was supplemented with 30% glycerol, aliquoted, flash frozen in an isopropanol bath, and stored at -80 °C.

[0211] Fluorescent labeling of proteins. Stock solutions of FN3 and MTD4 were passed through a desalting spin column (Bio-rad) to remove β-mercaptoethanol. 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. Excess dye was removed by passing the reaction mixture through a desalting spin column. The stoichiometry of dye labeling was measured on a Nanodrop spectrophotometer at 280 nm (λ of protein). max ) and 544 nm (TMR λ max ) was estimated by comparing the absorbance at

[0212] Subcellular localization assay. The pYBA1132 plasmid containing a green fluorescent protein (GFP) tag was transformed into A. tumefaciens strain GV3101 (Yan, X., et al. Mol. Plant Breed 10, 371-379 (2012)) and then used to infiltrate N. benthamiana leaves. Two days after infiltration, MTD4-Rh or FN3-Rh (10 μM) was sprayed onto N. benthamiana leaves, and the treated leaves were incubated at 23 °C in the dark. Two hours after spraying, the protein solution on the surface of N. benthamiana leaves was washed three times with H2O2. Red and green fluorescence were observed using a confocal laser scanning microscope with excitation at 488 nm and 514 nm, respectively.

[0213] DAB staining. Tobacco leaves were immersed in 3,3'-diaminobenzidine (DAB) staining buffer (10 mM MES, pH 6.5 and 1 mg / mL DAB) for 18 h at 23 °C. The leaves were then transferred to 90% (v / v) ethanol at 65 °C until transparent.

[0214] 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 pathogenesis-related genes. Gene transcription levels were normalized to the expression of the reference gene NtActin. The primers used to detect the expression of NtHSR203, NtCHN50, and NtActin were as previously described (Zhang, C., et al. J Exp Bot 70, 5407-5421 (2019)). The experiment was performed in three biological replicates.

[0215] Bacterial inoculation. Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC- strain, deficient in type III secretion system, was used for bacterial inoculation assay as previously described with minor modifications (Zhang, X., et al., Plant Mol Biol 90, 19-31 (2016)). Bacteria were grown in King's medium containing 25 mg / L rifampicin at 28 °C. Fresh overnight bacterial cultures were collected, centrifuged at 4000 rpm for 5 min, and washed twice with 10 mM MgCl2. The OD600 of the suspension was determined spectrophotometrically and further diluted to 0.2 with 10 mM MgCl2 and infiltrated into 4-week-old N. tabacum leaves. Samples were taken at 0 and 3 dpi. Samples were ground in 10 mM MgCl2 and serially diluted in King's medium containing 25 mg / L rifampicin. After 2 days of expansion on the plates, the number of colonies formed was counted.

[0216] Fungal inoculation. Tomato plants were sterilized with 75% (v / v) ethanol and sprayed with 10 μM MTD4-HrpZ, HrpZ, or MTD4. Prior to inoculation with B. cinerea, tomato fruits were wounded with a small incision made with a sharp blade. B. cinerea was cultured on potato dextrose agar (PDA) medium at 28 °C in an incubator. B. cinerea mycelial blocks were placed on the wound site 24 h after spraying with the protein solution. Disease symptoms were assessed 3 dpi.

[0217] Statistical analysis. Multiple comparisons were performed using one-way analysis of variance followed by Dunnett's multiple comparison test using GraphPad Prism version 8.00 for Windows.

[0218] Example 2: MTD4-HrpZ for disease control in crop plants To test the application of MTD4-HrpZ for disease control in crop plants, tomato plants were used since it is one of the most popular vegetables in the United States. First, the optimal concentration of MTD4-HrpZ was determined on tomato plants that induces sufficient defense responses against the bacterial spot pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, but does not cause cell death. Tomato seedlings (variety OH88119) were grown in a growth chamber at 26 °C during the day and 22 °C at night with a 12-h light / dark cycle. Four-week-old plants were sprayed with different concentrations of MTD4-HrpZ, HrpZ, and MTD4, and 24 h later, inoculated with Pst. Disease symptoms were observed one week after inoculation. Spraying tomato plants with 1 μM MTD4-HrpZ, but not MTD4 or HrpZ, effectively inhibited the invasion of Pst (Figures 6A-6C).

[0219] Furthermore, the efficacy of MTD4-HrpZ was tested against Xanthomonas euvesicatoria pv. perforans (Xep), the causative agent of bacterial spot disease. Similar to the results with Pst, application of 1 μM MTD4-HrpZ strongly protected 4-week-old tomato plants against Xep infection. Furthermore, crude bacterial cell lysates derived from E. coli cells overproducing MTD4-HrpZ were also effective in protecting 4-week-old tomato plants against the bacterial pathogen. Three-fold dilutions of crude cell lysates protected tomato plants against Xep infection (Figures 7A-7D).

[0220] To determine whether MTD4-HrpZ promotes plant growth, tomato seedlings were sprayed twice (6 and 16 days after germination) with 0.5 μM MTD4-HrpZ. Treatment with MTD4-HrpZ increased plant height and fresh weight by 15–20% compared to controls treated with MTD4 or HrpZ (Figures 8 and 9A–9D).

[0221] Taken together, the above results clearly demonstrate that MTD4 significantly increases the entry of HrpZ into plant cells during foliar application, enhancing immunity against bacterial pathogens and promoting growth. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11]

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, the cargo moiety comprising a plant bioactive moiety; wherein at least one cell penetrating peptide motif is 3-10 amino acid residues in length and has at least three arginine and / or lysine residues; or The peptide, wherein at least one cell-penetrating peptide motif is 3-10 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 2-8 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 with SEQ ID NO:

118.

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

5. The peptide according to 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 according to 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 according to 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 any of the BC and the DE, CD, or FG loops has a cell penetrating peptide motif.

8. The peptide according to 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 according to 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. 11. The peptide of any one of claims 1 to 10, wherein the 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 according to any one of claims 1 to 11, wherein the cell-penetrating peptide motif is RRRWWW (SEQ ID NO: 104) or WWWRRR (SEQ ID NO: 105).

13. 13. The peptide of any one of claims 1 to 12, comprising: TGRRRRRWWWSKPI (SEQ ID NO:111); APWWWRRRRYY (SEQ ID NO:112); GGRRRRRWWWVQE (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. 14. The peptide of any of claims 1 to 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 to 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 by a side chain within the membrane translocation domain.

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

17. The peptide of any one of claims 1 to 16, wherein the peptide comprises SEQ ID NO: 126, 127, or 128.

18. 18. The peptide of any one of claims 1 to 17, wherein the plant bioactive moiety comprises synthetically derived or naturally occurring flagellin and flagellin-related polypeptides (including those conserved within the genus Bacillus), thionin, harpin proteins or polypeptides or harpin-like polypeptides, elongation factor Tu (EF-Tu), phytosulfokine (PSKα), root hair promoting polypeptides (RHPPs), hypersensitive response elicitor proteins or polypeptides, or any combination thereof.

19. 1. A composition for delivering a cargo moiety into a plant cell, comprising SEQ ID NO: 122 covalently attached to a cargo moiety, said cargo moiety comprising a plant bioactive moiety.

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

21. 21. The composition of any one of claims 19 to 20, wherein the plant bioactive moiety comprises synthetically derived or naturally occurring flagellin and flagellin-related polypeptides, thionin, harpin proteins or polypeptides or harpin-like polypeptides, elongation factor Tu (EF-Tu), phytosulfokine (PSKα), root hair promoting polypeptides (RHPPs), hypersensitive response elicitor proteins or polypeptides, or any combination thereof.

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

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

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

25. 22. A method of protecting a plant from biotic stress; stimulating seeds during germination; protecting a plant from abiotic stress; enhancing plant growth, yield, health, lifespan, productivity and / or vigor; conferring multiple disease resistances to a plant; or any combination thereof, comprising contacting the plant with a peptide according to any one of claims 1 to 18 or a composition according to any one of claims 19 to 21.

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

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