Composition and uses of a MTD4-n21 fusion protein

Abstract

Described are peptides including 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 includes a plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90% sequence identity with SEQ ID NO: 126. Also described are methods of delivering a cargo moiety into a plant cell comprising contacting the plant cell with the peptide as disclosed herein.

Description

[0001] Attorney Docket No. 103362-028WO1

[0002] COMPOSITION AND USES OF A MTD4-N21 FUSION PROTEIN

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant No. GM122459 awarded by National Institutes of Health and Grant No. 2154863 awarded by National Science Foundation. The government has certain rights in the invention.

[0005] CROSS-REFERENCE TO RELATED APPLICATION

[0006] This application claims priority to, and the benefit of U.S. Provisional Application 63 / 697,817, filed on September 23, 2024, the contents of which is hereby incorporated in its entirety.

[0007] REFERENCE TO SEQUENCE LISTING

[0008] The Sequence Listing submitted September 23, 2025, as a text filed named “103362_028W01_ST26” created September 21, 2025, and having a file size of 300,962 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0009] BACKGROUND

[0010] Although fertilizers and pesticides play a crucial role in modem agriculture, their adverse impact on the environment and animal / human health have recently inspired a rapid rise in organic farming. It is clear that organic agriculture with a ~$80 billion market will become an essential, innovative farming system that balances sustainability with food / ecosystem security and human health benefits (Reganold and Wachter, 2016). A promising environment-friendly innovation is the use of (i) biological defense activators that improve plant immunity against pathogens and insects, and (ii) biostimulants that enhance plant growth and tolerance to abiotic stresses. Many of the plant defense activators and biostimulants now being used in organic farming are proteins / peptides that trigger the appropriate signal transduction pathways and thereby stimulate defense and / or growth. A formidable challenge in using these peptides / proteins, however, is their poor penetration efficiency in foliar applications and seed treatments (Nadendla, S.R., et al., Carbohyd Polym 199, 11-19 (2018)).

[0011] Cell-penetrating peptides (CPPs) are short (typically 5-30 amino acids), cationic, amphipathic, or hydrophobic peptides that facilitate the cellular uptake of diverse cargo Attorney Docket No. 103362-028WO1 molecules by eukaryotic cells ( Bechara, C., et al., 2013, 587: 1693-1702; and Sagan 2013). CPPs were first discovered in the early 1990’s ( Derossi, D., et al., 1994, J Biol Chem 269: 10444-10450; Vives, E., et al., 1997, J Biol Chem 272: 16010-16017). Since then, nearly 2000 CPPs have been reported, the vast majority of which are linear peptides. Despite much effort in academia and industry, commercial applications of linear CPPs have largely been unsuccessful because linear CPPs are proteolytically unstable and exhibit low cytosolic entry efficiencies. Cyclic CPPs, which have vastly improved cell entry efficiencies and metabolic stabilities (Qian, Z., et al., 2016, Biochemistry 55: 2601-2612), were more recently reported. However, cyclic CPPs contain nonproteinogenic amino acids and must be chemically synthesized; the high cost of their production makes them economically nonviable for agricultural applications. To overcome this limitation, a family of membrane translocation domains (MTDs) was engineered, which are based on the human fibronectin type III (FN3) domain (~90 amino acids). The MTDs are proteolytically stable and can be recombinantly produced in high yields in Escherichia coli or other host systems. One of the MTDs, MTD4, has demonstrated excellent cytosolic entry efficiencies for animal (Pei and Bhat 2023) and plant cells (Wang et al. 2023).

[0012] The demand for biopesticides for plant disease control is increasing because of their environmental benefits. The biopesticide harpin is a glycine-rich, cysteine-free, and heatstable protein produced in nature by Gram-negative pathogenic bacteria ( Choi, M.-S., et al., 2013, Mol Plant Microbe Interact 26: 1115-1122 ("Choi et al. 2013")). Harpin proteins elicit hypersensitive response (HR) in plants, activate defense, and enhance plant growth and tolerance to drought; at high concentrations, they can also cause plant cell death ( Ji, Z. L., et al., 2020, Int J Mol Sci 22 ("Ji et al. 2020")). HrpN, secreted by Erwinia amylovora, was the first harpin elicitor discovered (Wei, Z.-M., et al., 1992, Science 257: 85-88). HrpN induces cell death and the production of reactive oxygen species (ROS) in host and non-host plants. Since the discovery of HrpN over 30 years ago, many other harpins have been characterized from various plant-pathogenic bacteria and shown to have similar biological effects to HrpN (Choi et al. 2013). For example, a fragment of harpin HpaGXooc from Xanthomonas oryzae pv. oryzicola increases the yield of rice (Chen, L., et al., 2008, Phytopathology 98: 792-802). Ectopic expression of the harpin-encoding gene hrfl from Xanthomonas oryzae pv. oryzae (Xoo) enhances the drought tolerance of rice (Shao, M., et al., 2008, Plant Biotechnol. J 6: 73-81). Ji et al., reported that a 21-aa peptide derived from Attorney Docket No. 103362-028WO1

[0013] Hpal of Abo (N21) promotes plant growth, disease resistance, and drought tolerance in several crop plants (Ji et al. 2020).

[0014] The unique characteristics of harpin proteins render them ideal defense activators and growth promoters of crops. In fact, Eden BioScience Corp, developed the harpin product Messenger for crop plants in the early 2000’ s. Although effective in some applications, the product has not been widely used in crop production. A major drawback is the low bioactivity of the harpin protein in plants, because it does not readily penetrate plant tissues in foliar applications (tested both in the field and in greenhouses).

[0015] The compositions and methods disclosed herein address these and other needs.

[0016] SUMMARY

[0017] Disclosed herein are compounds, compositions, methods for making and using such compounds and compositions. In one aspect, disclose are peptides including 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 includes a plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126, where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues; or where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least two arginine and / or lysine residues and at least one other cell penetrating peptide motif is from 2 to 8 amino acid residues in length and has at least two hydrophobic residues. Also disclosed are methods of delivering a plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126 into a plant cell including contacting the plant cell with the peptide as disclosed herein.

[0018] In some embodiments, the peptide can include SEQ ID NO: 127 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 127.

[0019] In some embodiments, the peptide can include SEQ ID NO: 184 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 184. Attorney Docket No. 103362-028WO1

[0020] 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.

[0021] BRIEF DESCRIPTION OF THE FIGURES

[0022] The accompanying figures, 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.

[0023] Fig. 1 is an image of a Coomassie blue-stained SDS-PAGE gel showing the elution profile of MTD4-N21 from a Ni-NTA column. FT, 8M urea flow-through fraction; El, 8M urea + 250 mM imidazole; E2, 8M urea + 500 mM imidazole; E3, 8M Urea + 1 M imidazole.

[0024] Figs. 2A-2B are images showing MTD4-N21 induces cell death at low concentrations on tomato plants. Four-week tomato plants (Cultivar OH88119) were sprayed with different concentrations of purified MTD4-N21 (Fig. 2A) and synthesized N21 (Fig. 2B). Photos were taken 6 days after protein spray.

[0025] Figs. 3A-3D are images and graphs showing that MTD4-N21 induces strong defense gene expression at low concentrations (0.6 and 0.8 pM) in 4-week-old tomato plants. Fig. 3A-3B show cell death induced by MTD4-N21 (Fig. 3 A) but not by N21 (Fig. 3B). Figs. 3C-3D shows gene expression of NAC (Fig. 3C) and OLP (Fig. 3D) in MTD4-N21 and N21 -treated plants at 48 hr after protein spray. TWC: Tween / water control.

[0026] Figs. 4A-4F are images showing that MTD4-N21 significantly enhances tomato resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Four- week-old tomato plants were infiltrated with DC3000 with the vacuum method. Photos of the infected leaves in Fig. 4A-4D were taken 7 days after inoculation. Leaves of Figs. 4E-4F were destained to see the infection severity (black infection dots) in the control Fig. 4E and MTD4-N21 -treated leaves Fig. 4F.

[0027] Figs. 5A-5B are graphs showing that MTD4-N21 induces defense gene expression after Pst DC3000 infection. Tomato plants were inoculated with DC3000 at two days after spraying with MTD4-N21. Leaves were collected at 0, 12, 24 and 48 h after inoculation. Significant differences in gene expression levels of NAC (Fig. 5B) and LPSE (Fig. 5A) between MTD4-N21 and Tween / water control were observed 4 days after inoculation. NAC: plant-specific NAC-type transcription factor; LPSE: Lipid particle serine esterase. TWC: Tween / water control. Attorney Docket No. 103362-028WO1

[0028] Figs. 6A-6D are graphs showing that MTD4-N21 promotes plant growth of tomatoes in the growth chamber. Tomato plants at 3- and 5-week-old stages were sprayed with 0.5 and 0.7 pM MTD4-N21, respectively. 0.03% Tween20 / water (TW Con) was used as the control. Plant growth parameters were measured 7 weeks after germination. Significance has been counted as student t-test against TW control; test; *p < 0.05; ** p < 0.01; *** p < 0.001). (6A) shows plant fresh weight; (6B) plant dry weight; (6C) root fresh weight; and (6D) root dry weight.

[0029] Figs. 7A-7D are images showing that MTD4-N21 is stable up to 5 days at room temperature (RT). MTD4-N2 solutions (#31, 0.8 pM) freshly prepared, 1, 3 and 5 days kept at RT were used for the protein stability assay. Number of small and big cell death lesions in each treated plant were recorded 5 days after protein spray. Photos were taken 5 days after protein spray with MTD4-N2 solutions (#31, 0.8 pM) freshly prepared (Fig. 7A), 1 day (Fig. 7B), 3 days (Fig. 7C) and 5 days (Fig. 7D) kept at RT.

[0030] Fig. 8 shows the sequence alignment of bison and human FN3 domains. The three nonidentical amino acids are underlined.

[0031] Fig. 9 shows coomassie blue-stained SDS-PAGE gel showing the purity of lyophilized inclusion bodies of bMTD4-N21 (lane 2).

[0032] Figs. 10A-10D show that bMTD4-N21 induces cell death in tomato leaves. Four- week-old tomato (Solarium lycopersicum) plants were sprayed with purified bMTD4-N21 protein at concentrations of 2 (10B), 10 (10C), or 30 pM (10D), while 0.03% Tween was used as a control (10A). Plants were maintained under controlled conditions, and representative images were taken 5 days post-treatment.

[0033] Figs. 11 A-l ID show that bMTD4-N21 upregulates defense- and hypersensitive response-related genes in tomato leaves. Tomato plants treated with bMTD4-N21 protein exhibited a pronounced induction of defense and hypersensitive response (HR) marker genes. The expression profiles of selected genes, including NAC (NAC transcription factor) (11 A), LPSE (lipid particle serine esterase) (11C), OLP (Osmotin-like protein) (1 IB), and ACS2 (1-aminocyclopropane-l-carboxylate synthase) (HD), were analyzed by quantitative real-time PCR at 0 h and 2 days post-spray. Plants were treated with different concentrations of bMTD4-N21 protein (2, 10, or 30 pM), while 0.03% Tween served as a control. Gene expression levels were normalized relative to internal reference genes, and fold changes were calculated in comparison to the control treatment. Data are presented as mean ± SD. Different letters above the bars (A, B, C) indicate statistically significant Attorney Docket No. 103362-028WO1 differences within treatments, as determined by Tukey’s multiple comparison test (p 0.05). Gray color and light blue color, mentioning 0 h and 48 h, respectively.

[0034] DETAILED DESCRIPTION

[0035] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0036] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects 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, example methods and materials are now described.

[0037] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0038] Definitions

[0039] Throughout the present 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 those values near to a recited value, as well as the recited value.

[0040] Throughout the present specification, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise all values and subranges therein. Thus, the range “from 50 to 80” includes all possible values therein (e.g., 50, 51, 52, 53, 54, 55, 56, etc.) and all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55- 80, 50-75, etc.).

[0041] 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 Attorney Docket No. 103362-028WO1 polypeptide conjugate. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein. In addition, reference to “a polypeptide conjugate” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the polypeptide conjugates is present, unless the context clearly requires that there is one and only one of the polypeptide conjugates.

[0042] As used herein, the term “adjacent” refers to two contiguous amino acids, which are connected by a covalent bond. “Adjacent” is also used interchangeably with “consecutive.”

[0043] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0044] As used herein, “cell penetrating peptide” or “CPP” refers to any peptide including proteins (i.e., polypeptides) which is capable of penetrating a cell membrane. As used herein, “cyclic cell penetrating peptide” or “cCPP” refers to any cyclic peptide which is capable of penetrating a cell membrane.

[0045] A “foliar treatment” as used herein refers to a composition that is applied to the above ground parts or foliage of a plant or plant part and may have leaves, stems, flowers, branches, or any aerial plant part, for example, scion.

[0046] As used herein, “linker” or “L” refers to a moiety that covalently attaches two or more components of the polypeptide conjugates disclosed herein (e.g., a linker may covalently attach a CPP and a group that binds to a nucleic acid sequence by electrostatic interactions (i.e., P). In some embodiments, the linker can be natural or non-natural amino acid or polypeptide. In other embodiments, the linker is a synthetic compound containing two or more appropriate functional groups suitable to bind, e.g., the CPP and, independently, P. In some embodiments, the linker is about 3 to about 100 (e.g., about 3 to about 20) atoms in linear length (not counting the branched atoms or substituents). In some embodiments, the linker provides about 1 A to about 400 A in distance of the two groups to which it connects.

[0047] As used herein, “polypeptide” refers to a string of at least two amino acids attached to one another by a peptide bond. There is no upper limit to the number of amino acids that can be included in a polypeptide. Further, polypeptides may include non-natural amino acids, Attorney Docket No. 103362-028WO1 amino acid analogs, or other synthetic molecules that are capable of integrating into a polypeptide.

[0048] As used herein, a “monomer” refers to an amino acid residue in a polypeptide. In some embodiments, an amino acid monomer is divalent. In other embodiments, an amino acid monomer may be trivalent if the monomer is further substituted. For example, a cysteine monomer can independently form peptide bonds at the N and C termini, and also form a disulfide bond.

[0049] As used herein, an “amino acid-analog” or “analog” (e.g., “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 an oligonucleotide through electrostatic interactions. Such variants may have an elongated or shorter side chain (e.g., by one or more -CH2- groups that retains the ability to bind an oligonucleotide through electrostatic interactions, or alternatively, the modification can improve the ability to bind an oligonucleotide through electrostatic interactions. For example, an arginine analog may include an additional methylene or ethylene between the backbone and guanidine / guanidinium group. Other examples include amino acids with one or more additional substituents (e.g., Me, Et, halogen, thiol, methoxy, ethoxy, Cl-haloalkyl, C2- haloalkyl, amine, guanidine, etc). The amino acid-analog can be monovalent, divalent, or trivalent.

[0050] Throughout the present specification, peptides and amino acid monomers are depicted as charge neutral species. It is to be understood that such species may bear 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 an amino acid is deprotonated and bears a negative charge (-CO2 ). Similarly, the side chains of certain amino acids may bear a positive or negative charge.

[0051] Each amino acid can be a natural or non-natural amino acid. The term “non-natural amino acid” refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid can be a modified amino acid, and / or amino acid analog, that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be the D-isomer of the natural amino acids. Thus, as used herein, the term “amino acid” refers to natural and non-natural amino acids, and analogs and derivatives thereof. Examples of Attorney Docket No. 103362-028WO1 suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative, or combinations thereof. Analogs of amino acids encompass that have a structural similar but not identical to an amino acid, e.g., due to a modification to the side chain or backbone on said amino acid. Such modifications may increase the hydrophobicity of the side chain, including elongation of the side chain by one or more hydrocarbons, or increasing the the solvent accessible surface area (SASA as described herein) of an amino acid having an aromatic ring on its side chain, e.g., by conjugating a second aromatic ring or increasing the size of the aromatic ring. Derivatives of amino acids encompass natural and non-natural amino acids that have been modified (e.g., by susbstitution) to include a hydrophobic group as described herein. For example, a derivative of lysine includes lysine whose side chain has been substituted with alkylcarboxamidyl. These, and others, are listed in the Table 1 along with their abbreviations used herein.

[0052] Table 1. Amino Acid Abbreviations Attorney Docket No. 103362-028WO1

[0053] * single letter abbreviations: when shown in capital letters herein it indicates the L-amino acid form, when shown in lower case herein it indicates the D-amino acid form.

[0054] A “variant” refers to a molecule substantially similar in structure. Thus, in one embodiment, a variant refers to a protein whose amino acid sequence is similar to a reference amino acid sequence, but does not have 100% identity with the respective Attorney Docket No. 103362-028WO1 reference sequence. The variant protein has an altered sequence in which one or more of the amino acids in the reference sequence is deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the variant protein has an amino acid sequence which is at least 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the reference sequence. For example, variant sequences which are at least 95% identical have no more than 5 alterations, i.e. any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence.

[0055] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full- length of the sequences being compared can be determined by known methods. Attorney Docket No. 103362-028WO1

[0056] For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0057] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positivevalued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. Attorney Docket No. 103362-028WO1

[0058] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- 5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0059] “Alkyl” or “alkyl group” refers to a fully saturated, straight or branched hydrocarbon chain radical having from one to twelve carbon atoms, and which is attached to the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms from 1 to 12 are included. An alkyl comprising up to 12 carbon atoms is a C1-C12 alkyl, an alkyl comprising up to 10 carbon atoms is a C1-C10 alkyl, an alkyl comprising up to 6 carbon atoms is a Ci-Ce alkyl and an alkyl comprising up to 5 carbon atoms is a C1-C5 alkyl. A C1-C5 alkyl includes C5 alkyls, C4 alkyls, C3 alkyls, C2 alkyls and Ci alkyl (z.e., methyl). A Ci-Ce alkyl includes all moieties described above for C1-C5 alkyls but also includes C& alkyls. A C1-C10 alkyl includes all moieties described above for C1-C5 alkyls and Ci-Ce alkyls, but also includes C7, Cs, C9 and C10 alkyls. Similarly, a C1-C12 alkyl includes all the foregoing moieties, but also includes Cn and C12 alkyls. Non-limiting examples of C1-C12 alkyl include methyl, ethyl, zz-propyl, z-propyl, ec-propyl, zz-butyl, z-butyl, sec-butyl, / -butyl, zz-pentyl, Z-amyl, zz-hexyl, zz-heptyl, zz-octyl, zz-nonyl, zz-decyl, zz- undecyl, and zz-dodecyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0060] “Alkylene” or “alkylene chain” refers to a fully saturated, straight or branched divalent hydrocarbon chain radical, having from one to forty carbon atoms. Non-limiting examples of C2-C40 alkylene include ethylene, propylene, zz-butylene, pentylene, and the like. Unless stated otherwise specifically in the specification, an alkylene chain can be optionally substituted as described herein.

[0061] “Alkenyl” or “alkenyl group” refers to a straight or branched hydrocarbon chain radical having from two to twelve carbon atoms, and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl group comprising any number of carbon atoms from 2 to 12 are included. An alkenyl group comprising up to 12 carbon atoms is a C2-C12 alkenyl, an alkenyl comprising Attorney Docket No. 103362-028WO1 up to 10 carbon atoms is a C2-C10 alkenyl, an alkenyl group comprising up to 6 carbon atoms is a C2-C6 alkenyl and an alkenyl comprising up to 5 carbon atoms is a C2-C5 alkenyl. A C2-C5 alkenyl includes C5 alkenyls, C4 alkenyls, C3 alkenyls, and C2 alkenyls. A C2-C6 alkenyl includes all moieties described above for C2-C5 alkenyls but also includes Ce alkenyls. A C2-C10 alkenyl includes all moieties described above for C2-C5 alkenyls and C2- Ce alkenyls, but also includes C7, Cs, C9 and C10 alkenyls. Similarly, a C2-C12 alkenyl includes all the foregoing moieties, but also includes Cn and C12 alkenyls. Non-limiting examples of C2-C12 alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-l -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 specifically in the specification, an alkyl group can be optionally substituted.

[0062] “Alkenylene” or “alkenylene chain” refers to a straight or branched divalent hydrocarbon chain radical, having from two to forty carbon atoms, and having one or more carbon-carbon double bonds. Non-limiting examples of C2-C40 alkenylene include ethenylene (-CH=CH-), propenylene, butenylene, and the like. Unless stated otherwise specifically in the specification, an alkenylene chain can be optionally substituted.

[0063] “Alkynyl” or “alkynyl group” refers to a straight or branched hydrocarbon chain radical having from two to twelve carbon atoms and having one or more carbon-carbon triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl group comprising any number of carbon atoms from 2 to 12 are included. An alkynyl group comprising up to 12 carbon atoms is a C2-C12 alkynyl, an alkynyl comprising up to 10 carbon atoms is a C2-C10 alkynyl, an alkynyl group comprising up to 6 carbon atoms is a C2-C6 alkynyl and an alkynyl comprising up to 5 carbon atoms is a C2-C5 alkynyl. A C2-C5 alkynyl includes C5 alkynyls, C4 alkynyls, C3 alkynyls, and C2 alkynyls. A C2-C6 alkynyl includes all moieties described above for C2-C5 alkynyls but also includes Ce Attorney Docket No. 103362-028WO1 alkynyls. A C2-C10 alkynyl includes all moieties described above for C2-C5 alkynyls and C2- Ce alkynyls, but also includes C7, Cs, C9 and C10 alkynyls. Similarly, a C2-C12 alkynyl includes all the foregoing moieties, but also includes Cn and C12 alkynyls. Non-limiting examples of C2-C12 alkenyl include ethynyl, propynyl, butynyl, pentynyl and the like. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0064] “Alkynylene” or “alkynylene chain” refers to a straight or branched divalent hydrocarbon chain radical, having from two to forty carbon atoms, and having one or more carbon-carbon triple bonds. Non-limiting examples of C2-C40 alkynylene include ethynylene (-C=C-), propargylene and the like. Unless stated otherwise specifically in the specification, an alkynylene chain can be optionally substituted.

[0065] “Aryl” refers to a hydrocarbon ring system comprising hydrogen, 6 to 40 carbon atoms and at least one aromatic ring. For purposes of this disclosure, the aryl can be a monovalent or a divalent radical (not counting substituents), which can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and which 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, a.s-indacene, -indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. In some embodiments, the aryl radical can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, an aryl group can be optionally substituted.

[0066] As used herein “aromatic” refers to an unsaturated cyclic molecule having 4n + 27t electrons, wherein n is any integer. The term “non-aromatic” refers to any unsaturated cyclic molecule which does not fall within the definition of aromatic.

[0067] “Carbocyclyl,” “carbocyclic ring” or “carbocycle” refers to a rings structure, wherein the atoms which form the ring are each carbon. Carbocyclic rings can comprise from 3 to 20 carbon atoms in the ring. Carbocyclic rings include aryls and cycloalkyl and rings that are fully unsaturated, partially unsaturated, and fully saturated. In some embodiments, the carbocyclyl can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, a carbocyclyl group can be optionally substituted.

[0068] “Cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon radical having from 3 to 40 carbon atoms and at least one ring, wherein the Attorney Docket No. 103362-028WO1 ring consists solely of carbon and hydrogen atoms, which can include fused or bridged ring systems. For purposes of this disclosure, the cycloalkyl can be a monovalent or a divalent radical (not counting substituents). Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbomyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. In some embodiments, the cycloalkyl radical can be divalent when used as a linker or as a part of a linker. Unless otherwise stated specifically in the specification, a cycloalkyl group can be optionally substituted.

[0069] “Cycloalkenyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having from 3 to 40 carbon atoms, at least one ring having, and one or more carbon-carbon double bonds, wherein the ring consists solely of carbon and hydrogen atoms, which can include fused or bridged ring systems. For purposes of this invention, the cycloalkenyl can be a monovalent or a divalent radical (not counting substituents). Monocyclic cycloalkenyl radicals include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycloctenyl, and the like. Polycyclic cycloalkenyl radicals include, for example, bicyclo[2.2.1]hept-2-enyl and the like. In some embodiments, the cycloalkenyl radical can be divalent when used as a linker or as a part of a linker. Unless otherwise stated specifically in the specification, a cycloalkenyl group can be optionally substituted.

[0070] “Cycloalkynyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having from 3 to 40 carbon atoms, at least one ring, and one or more carbon-carbon triple bonds, wherein the ring consists solely of carbon and hydrogen atoms, which can include fused or bridged ring systems. For purposes of this invention, the cycloalkynyl can be a monovalent or a divalent radical (not counting substituents). Monocyclic cycloalkynyl radicals include, for example, cycloheptynyl, cyclooctynyl, and the like. In some embodiments, the cycloalkynyl radical can be divalent when used as a linker or as a part of a linker. Unless otherwise stated specifically in the specification, a cycloalkynyl group can be optionally substituted.

[0071] “Heterocyclyl,” “heterocyclic ring” or “heterocycle” refers to a stable 3- to 20-membered aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this invention, the heterocyclyl radical can be a monovalent or a divalent radical (not counting substituents). Heterocyclycl or heterocyclic rings include heteroaryls as defined below. Unless stated otherwise specifically in the specification, the heterocyclyl Attorney Docket No. 103362-028WO1 radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and 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[l,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, the heterocyclyl radical can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, a heterocyclyl group can be optionally substituted.

[0072] “Heteroaryl” refers to a 5- to 20-membered ring system radical comprising hydrogen atoms, one to fourteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of this invention, the heteroaryl radical can be a monovalent or a divalent radical (not counting substituents) and can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodi oxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[Z>][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotri azolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1 -phenyl- U7-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, Attorney Docket No. 103362-028WO1 triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). In some embodiments, the heteroaryl radical can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, a heteroaryl group can be optionally substituted.

[0073] The term “ether” used herein refers to a straight or branched divalent radical moiety -[(CH2)m-O-(CH2)n]z- wherein each of m, n, and z are independently selected from 1 to 40. Examples include, but are not limited to, polyethylene glycol. Unless stated otherwise specifically in the specification, the ether can be optionally substituted.

[0074] The term “substituted” used herein means any of the above groups (i.e., alkylene, alkenylene, alkynylene, aryl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, and / or ether) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, “substituted” includes any of the above groups in which one or more hydrogen atoms are replaced with -NRgRh, -NRgC(=O)Rh, -NRgC(=O)NRgRh, -NRgC(=O)ORh, -NRgSO2Rh, -OC(=O)NRgRh, -ORg, -SRg, -SORg, -SO2Rg, -OSO2Rg, -SO2ORg, =NSO2Rg, and -SO2NRgRh. “Substituted also means any of the above groups in which one or more hydrogen atoms are replaced with -C(=O)Rg, -C(=O)ORg, -C(=O)NRgRh, -CIESChRg, -CEESChNRgRh. In the foregoing, Rgand Rh are the same or different and independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, A-heterocyclyl, heterocyclylalkyl, heteroaryl, A-heteroaryl and / or heteroarylalkyl. “Substituted” further means any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, Attorney Docket No. 103362-028WO1 alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, A-heterocyclyl, heterocyclylalkyl, heteroaryl, A-heteroaryl and / or heteroarylalkyl group. In addition, each of the foregoing substituents can also be optionally substituted with one or more of the above substituents. Further, those skilled in the art will recognize that “substituted” also encompasses instances in which one or more atoms on any of the above groups are replaced by a substituent listed in this paragraph, and the substituent forms a covalent bond with the CPP, P, or L. For example, in certain embodiments, any of the above groups can be substituted at a first position with a carboxylic acid (i.e., -C(=O)OH) which forms an amide bond with a lysine in the CPP, or a group can be substituted at a second position with a thiol group which forms a disulfide bond with a cysteine (or amino acid analog having a thiol group).

[0075] A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained 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.

[0076] As used herein, the symbol “ ” (hereinafter can be referred to as “a point of attachment bond”) denotes a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other of which is not depicted as being attached to the point of attachment bond. For example, ” indicates that the chemical entity “XY” is bonded to another chemical entity via the point of attachment bond. Furthermore, the specific point of attachment to the non-depicted chemical entity can be specified by inference. For example, the compound

[0077] XY-|-

[0078] CH3-R3, wherein R3is H or “ ” infers that when R3is “XY”, the point of attachment bond is the same bond as the bond by which R3is depicted as being bonded to CH3.

[0079] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, Attorney Docket No. 103362-028WO1 thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the compounds and compositions disclosed herein include all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[0080] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about 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 (-) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non- superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0081] Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically- labeled or isotopically-substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of Attorney Docket No. 103362-028WO1 isotopes that can be incorporated into compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,170,35S,18F and36C1 respectively. Compounds further comprise prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e.,3H, and carbon-14, i.e.,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.

[0082] Disclosed are the components to be used to prepare the compositions disclosed herein as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of 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 methods of making and using the compositions disclosed herein. Thus, if there are a variety Attorney Docket No. 103362-028WO1 of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods disclosed herein.

[0083] The term "contacting" as used herein refers to bringing a disclosed compound and a target (e.g., a cell, target receptor, transcription factor, or other biological entity) together in such a manner that the compound can affect 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 is dependent.

[0084] As used herein, the terms "effective amount" and "amount effective" refer to an amount that is sufficient to achieve the desired result.

[0085] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

[0086] Compounds

[0087] Disclosed are cell-permeable peptides and compositions comprising them, which can provide a general vehicle for cytosolic delivery of potentially any peptide or protein cargo as well as other biomolecules including oligonucleotides. The disclosed peptides can have greater cytosolic delivery efficiency and in vivo stability over simple cell permeable peptides.

[0088] In a specific aspect, disclosed herein are peptides comprising: a membrane translocation domain having one or more cell penetrating peptide motifs, and a cargo moiety linked to the membrane translocation domain, wherein the cargo moiety includes a plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126, where at least one of the cell penetrating peptide motifs is from 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues.

[0089] In some embodiments, the peptide can include SEQ ID NO.s: 119, 120, 121, 122, 123, 124, 125, 127, 167, 168, 169, 170, 171, or 184 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity. Attorney Docket No. 103362-028WO1

[0090] In some embodiments, the peptide can include SEQ ID NO: 127 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 127.

[0091] In some embodiments, the peptide can include SEQ ID NO: 184 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 184.

[0092] Unlike methods where a CPP motif is inserted into each target protein, the disclosed compounds, compositions and methods involve an engineered membrane translocation domain that can be genetically or synthetically fused to any target cargo of interest. Another strategy disclosed herein involves splitting a CPP motif into two halves and inserting them into two different regions of the membrane translocation domain, resulting in greatly improved cytosolic delivery efficiencies.

[0093] In some embodiments, the compounds described herein can be used as plant activators. The term “plant activator” refers to a compound that activates a natural defense mechanism in a host plant, such as systemic acquired resistance (SAR), or hypersensitive response. The compositions can be used as a plant activators for either healthy and unhealthy plants, or plants in both healthy and unhealthy environments.

[0094] In some embodiments, the compounds described herein can be used as plant stimulants. The term “plant stimulant,” as used herein refers to a compound or compositions applied to plants under conditions that enhance nutrition efficiency, stress tolerance, and / or crop quality traits, regardless of its nutrition content. Particularly, plant stimulants are used in the cultivation of plants in order to improve the growth and development processes. The impact of stimulants on plants is not due to direct participation in the regulation of life processes, but the effect on metabolism in the broad sense of this word. They can stimulate the synthesis of natural hormones, and sometimes increase their activity, can improve intake of minerals from the soil, regulate the growth of roots. In addition, they can cause the increase of the resistance to adverse conditions (biotic or abiotic). The use of stimulants in the cultivation of plants increases the yields, often while increasing their quality at the same time. Stimulants can enhance life processes occurring in plants without changing plants natural behavior. The compounds and / or compositions described herein can be plant stimulants and therefore can be used as plant growth regulators, plant metabolic processes regulators, plant physiological processes regulators, a substance that prevents against the effects of biotic or abiotic stress in a plant, and / or a substance that provides multiple disease resistance to a plant. Attorney Docket No. 103362-028WO1

[0095] The compositions can be used as a plant stimulant for either healthy and unhealthy plants, or plants in both healthy and unhealthy environments.

[0096] Membrane Translocation Domain

[0097] The membrane translocation domain portion of the disclosed peptides can be any membrane translocation domain, a peptide sequence that may traverse a lipid bilayer, that has been modified to contain at least one cell penetrating motifs as described herein. In a preferred example, there are two or three cell penetrating motifs in the membrane translocation domains. For example, at least one cell penetrating peptide motif can be from 3 to 10 amino acid residues in length and have at least three arginine and / or lysine residues, e.g., 4, 5, or 6 arginines and / or lysine residues. Alternatively, at least one cell penetrating peptide motif can be from 3 to 10 amino acid residues in length and have at least two arginine and / or lysine residues and at least one other cell penetrating peptide motif can be from 2 to 8 amino acid residues in length and have at least two hydrophobic residues. When there are two or more cell penetrating peptide motifs, there can be two or more arginine residues and / or lysine residues in a 3 to 10 amino acid span and another cell penetrating peptide motif where there are two or more hydrophobic residues within a 2 to 8 amino acid span. The cell penetrating peptide motifs can be anywhere in the membrane translocation domain.

[0098] 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 a specific example, the membrane translocation domain has at least 90%, at least 95%, or at least 97% sequence similarity with SEQ. ID. NO. : 118. In other examples, the membrane translocation domain is 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, e.g., the cell penetrating peptide motif is in two of the BC, DE, CD, or FG loops, in particular 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).

[0099] In other examples, the membrane translocation domain can be any stably folded protein, which can preferably be efficiently expressed in bacteria. Some additional examples of membrane translocation domains are the nanobody scaffold, DARPin scaffold, Attorney Docket No. 103362-028WO1 and CTPR protein (the consensus tetratricopeptide repeat; Acc. Chem. Res. 2021, 54, 4166-4177).

[0100] Cell Penetrating Peptide Motif

[0101] The cell penetrating peptide (CPP) motif can comprises at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, or at least 6 amino acids, more specifically from 3 to 8, from 3 to 6, from 4 to 8, from 4 to 6, or from 6 to 8 amino acids. In most examples, the CPP motif is substituted into the membrane translocation domain such that the resulting peptide has the same number of amino acids as in native membrane translocation domain.

[0102] In some examples, at least two, three, four, five, six, or seven amino acids of the CPP motif are adjacent arginine residues. In a preferred, example there are three, four, or five adjacent arginine residues in a CPP motif. In other examples, the arginie residues are not adjacent in the CPP motif. Each amino acid in the CPP motif can independently be a natural or non-natural amino acid. When such adjacent arginine or lysine residues are the CPP motif, then there need not be any additional CPP motifs, e.g., those with hydrophobic residues, though such a hydrophobic CPP motif can still be used. When the CPP motif contains two argine residues, then it is preferred that there be another CPP motif with at least two hydrophobic residues within 2 to 8 amino acids.

[0103] In other examples, at least one, at least, two, at least three, or more amino acids of the CPP motif are hydrophobic amino acids, i.e., have hydrophobic side chains. In some examples, the amino acids 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- methylbenzyljcysteine, N-(naphthalen-2-yl)glutamine, 3-(l,l'-biphenyl-4-yl)-alanine, tertleucine, or nicotinoyl lysine, each of which is optionally substituted with one or more substituents. In particular examples, 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 acids having hydrophobic side chains are phenylalanine, naphthylalanine, tryptophan, or an analog or derivative thereof naphthylalanine or tryptophan, or analogues or derivatives Attorney Docket No. 103362-028WO1 thereof. In other examples, the CPP motif further comprises at least one phenylalanine, phenylglycine, or histidine, or analogues or derivatives thereof.

[0104] 3-(2-quinolyl)-alanine O-benzylserine 3-(4-(benzyloxy)phenyl)-alanine

[0105] 3-(3-benzothienyl)-alanine

[0106] In some examples, the CPP motif can include any combination of at least three adjacent arginines and either at least two amino acids have a hydrophobic side chain selected from an aryl or heteroaryl, wherein the aryl and heteroaryl are optionally substituted, with a total number of amino acids in the CPP motif in the range of from 5 to about 8 amino acids.

[0107] 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 a specific example, the CPP motif is in the BC and either the DE, CD and FG loops, preferably in the BC and FG loops.

[0108] Where there are two or more CPP motifs, one CPP motif can be the 3 to 10 amino acid segment with at least two arginine and / or lysine residues and the other can be a 2 to 8 amino acid segment with at least two hydrophobic residues. For example, the membrane translocation domain can have two or more CPPs and at least one of the motifs is from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues. Attorney Docket No. 103362-028WO1

[0109] In an example of this, the membrane translocation domain can be human fibronectin type III having BC, DE, CD, and FG loops, and the CPP motifs can be in the BC loop and have from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues and a CPP motif can be in the FG loop and have from 3 to 10 amino acid residues and has at least three adjacent arginine and / or lysine residues. Alternatively, the CPP motifs can be in the FG loop and have from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues and a CPP motif can be in the BC loop and have from 3 to 10 amino acid residues and has at least three adjacent arginine and / or lysine residues.

[0110] When the CPP motif contains the from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, it can be WW, FF, WF, FW, WWW, FFF, WFW, FWF, WWF, WFF, FWW, FFW, WYW, WWH, YWW, or WYH. It is preferable that this CPP motif be in the BC loop. It is further preferable that this CPP motif be WW, FW, WF, WYW, WWW, WWH, YWW, WYH or YWH.

[0111] The CPP motif with 3 to 10 amino acid residues and has at least three adjacent arginine and / or lysine residues can contain RRR, RRRR (SEQ. ID. NO. 181), RRRRR (SEQ. ID. NO. 182). It can also be any combination of arginine and lysine residues. When this CPP motif is in the FG loop I can be 3-10 residues in length and of any combinations of Arg and Lys (and occasionally other non-acidic residues). The CPP motif (e.g., WWWRRRR (SEQ. ID. NO. 183)) may be alternatively split, so that some of the Arg / Lys residues are moved from the FG loop into the BC loop (e.g., WWWR. . .RRR, WWWRR. . RR, WWWRRRR. . . , etc ); The CPP motif (e g., WWWRRRR) may be alternatively split, so 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, so that either BC or FG loop contains a combination of hydrophobic and positively charged residues (e.g., WWR. . . WRRR, WWRR. . . WRR, WWRR. . RRW, RRW. . . WWRR, etc ).

[0112] In specific examples, the CPP motif comprises SEQ. ID. NOS.: 104, 105, 111, 112, 113, 114, 115, 116, or 117.

[0113] In some examples, the CPP motif can be or comprise any of the sequences listed in Table 2. In some examples, the cell penetrating peptide can be or comprise the reverse of any of the sequences listed in Table 2. Attorney Docket No. 103362-028WO1

[0114] Table 2. CPP motif sequences Attorney Docket No. 103362-028WO1 Attorney Docket No. 103362-028WO1 = L-naphthylalanine; (|) = D-naphthylalanine; Q = L-norleucine; r = D-arginine; F = L- phenylalanine; f = D-phenylalanine; q = D-glutamine; X = L-4-fluorophenylalanine; Dap = L-2,3-diaminopropionic acid; Sar, sarcosine; F2Pmp, L-difluorophosphonomethyl phenylalanine; Dod, dodecanoyl; Pra, L-propargylglycine; AzK, L-6-Azido-2-amino- hexanoic; Agp, L-2-amino-3-guanidinylpropionic acid; ^Cyclization between Pirn and Nlys;cCyclization between Lys and Glu; "'Macrocyclization by multicomponent reaction with aziridine aldehyde and isocyanide; ^Cyclization between the main-chain of Gin residue; 'N- terminal amine and side chains of two Dap residues bicyclized with Tm;gThree Cys side chains bicyclized with tris(bromomethyl)benzene; ^Cyclization by the click reaction between Pra and Azk.

[0115] The chirality of the amino acids can be selected to improve cytosolic uptake efficiency. In some embodiments, at least two of the amino acids have the opposite chirality. In some embodiments, the at least two amino acids having the opposite chirality can be adjacent to each other. In some embodiments, at least three amino acids have alternating stereochemistry relative to each other. In some embodiments, the at least three amino acids having the alternating chirality relative to each other can be adjacent to each other. In some embodiments, at least two of the amino acids have the same chirality. In some embodiments, the at least two amino acids having the same chirality can be adjacent to each other. In some embodiments, at least two amino acids have the same chirality and at least two amino acids have the opposite chirality. In some embodiments, the at least two Attorney Docket No. 103362-028WO1 amino acids having the opposite chirality can be adjacent to the at least two amino acids having the same chirality. Accordingly, in some embodiments, adjacent amino acids in the cCPP can have any of the following sequences: D-L; L-D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D.

[0116] Cargo moiety

[0117] The cargo moiety can be linked to the membrane translocation domain. The cargo moiety can be linked to an amino group (e.g., N-terminus), a carboxylate group (e.g., C- terminus), or a side chain of one or more amino acids in the in the membrane translocation domain.

[0118] When the cargo moiety is attached to the side chain of an amino acid in the membrane translocation domain, the membrane translocation domain includes an amino acid having a side chain with a suitable functional group to form a covalent bond (conjugation) with the cargo, or a side chain which may be modified to provide a suitable functional group (e.g., via conjugation of a linker) that forms a covalent bond with the cargo. In some embodiments, the amino acid on membrane translocation domain which has a side chain suitable conjugation of the cargo is a cysteine residue, glutamic acid residue, an aspartic acid residue, a lysine residue, or a 2,3 -diaminopropionic acid residue. In such embodiments, the cargo may be directly conjugated to the side chain of the amino acid (e.g., by forming a disulfide bond with a cysteine residue or an amide bond with a glutamic acid residue or a 2,3-diaminopropionic acid residue) or the cargo may be conjugated to the amino acid side chain through a linker (e.g., PEG).

[0119] In some embodiments, the cargo moiety can include a plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90%(e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126. In some embodiments, the cargo moiety can further include any cargo of interest, for example a linker moiety, a detectable moiety, or any combination thereof. In some examples, the cargo moiety can comprise one or more additional amino acids (e.g., K, UK, TRV); a linker (e.g., bifunctional linker LC-SMCC); coenzyme A; phosphocoumaryl amino propionic acid (pCAP); 8-amino-3,6-dioxaoctanoic acid (miniPEG); L-2,3-diaminopropionic acid (Dap or J); L-P-naphthylalanine; L-pipecolic acid (Pip); sarcosine; trimesic acid (Tm); 7-amino-4- methylcourmarin (Amc); fluorescein isothiocyanate (FITC); L-2-naphthylalanine; norleucine; 2-aminobutyric acid; Rhodamine B (Rho); Dexamethasone (DEX); or combinations thereof. Attorney Docket No. 103362-028WO1

[0120] Plant bioactive moiety

[0121] The cargo moiety can include a plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90%(e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126. In some embodiments, a detectable moiety can be linked to a plant bioactive moiety. The plant bioactive moiety can be attached to the cell penetrating peptide moiety at the amino group, the carboxylate group, or the side chain of any of the amino acids of the cell penetrating peptide moiety (e.g., at the amino group, the carboxylate group, or the side chain or any of amino acid of the CPP). In some examples, the plant bioactive moiety can be attached to the detectable moiety.

[0122] The plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90%(e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126 can activates a natural defense mechanism in a host plant, such as systemic acquired resistance (SAR), or hypersensitive response; or enhance stress tolerance, and / or crop quality traits, regardless of its nutrition content.

[0123] Detectable moiety

[0124] The detectable moiety can comprise any detectable label. Examples of suitable detectable labels include, but are not limited to, a UV-Vis label, a near-infrared label, a luminescent group, a phosphorescent group, a magnetic spin resonance label, a photosensitizer, a photocleavable moiety, a chelating center, a heavy atom, a radioactive isotope, an isotope detectable spin resonance label, a paramagnetic moiety, a chromophore, or any combination thereof. In some embodiments, the label is detectable without the addition of further reagents.

[0125] In some embodiments, the detectable moiety is a biocompatible detectable moiety, such that the compounds can be suitable for use in a variety of biological applications. “Biocompatible” and “biologically compatible”, as used herein, generally refer to compounds that are, along with any metabolites or degradation products thereof, generally non-toxic to cells and tissues, and which do not cause any significant adverse effects to cells and tissues when cells and tissues are incubated (e.g., cultured) in their presence.

[0126] The detectable moiety can contain a luminophore such as a fluorescent label or nearinfrared label. Examples of suitable luminophores include, but are not limited to, metal porphyrins; benzoporphyrins; azabenzoporphyrine; napthoporphyrin; phthalocyanine; polycyclic aromatic hydrocarbons such as perylene, perylene diimine, pyrenes; azo dyes; xanthene dyes; boron dipyoromethene, aza-boron dipyoromethene, cyanine dyes, metal- Attorney Docket No. 103362-028WO1 ligand complex such as bipyridine, bipyridyls, phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium; acridine, oxazine derivatives such as benzophenoxazine; aza-annulene, squaraine; 8-hydroxyquinoline, polymethines, luminescent producing nanoparticle, such as quantum dots, nanocrystals; carbostyril; terbium complex; inorganic phosphor; ionophore such as crown ethers affiliated or derivatized dyes; or combinations thereof. Specific examples of suitable luminophores include, but are not limited to, Pd (II) octaethylporphyrin; Pt (Il)-octaethylporphyrin; Pd (II) tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II) meso-tetraphenylporphyrin tetrabenzoporphine; Pt (II) meso-tetrapheny metrylbenzoporphyrin; 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-l,10-phenanthroline) (Ru (dpp)s); Ru (II) tris(l,10-phenanthroline) (Ru(phen)s), tris(2,2’-bipyridine)rutheniurn (II) chloride hexahydrate (Ru(bpy)s); erythrosine B; fluorescein; fluorescein isothiocyanate (FITC); eosin; iridium (III) ((N- methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)); indium (III) ((benzothiazol-2-yl)- 7- (diethylamino)-coumarin))-2-(acetylacetonate); Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow; Texas Red; rhodamine B; rhodamine 6G; sulfur rhodamine; m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue; bromocresol green; bromcresol red; bromothymol blue; Cy2; a Cy3; a Cy5; a Cy5.5; Cy7; 4- nitirophenol; alizarin; phenolphthalein; o-cresolphthalein; chlorophenol red; calmagite; bromo-xylenol; phenol red; neutral red; nitrazine; 3,4,5,6-tetrabromphenolphtalein; congo red; fluorescein; eosin; 2',7'-dichlorofluorescein; 5(6)-carboxy-fluorecsein; carboxynaphthofluorescein; 8-hydroxypyrene-l,3,6-trisulfonic acid; semi- naphthorhodafluor; semi -naphthofluorescein; tris (4,7-diphenyl-l,10-phenanthroline) ruthenium (II) di chloride; (4,7-diphenyl-l,10-phenanthroline) ruthenium (II) tetraphenylboron; platinum (II) octaethylporphyin; dialkylcarbocyanine; dioctadecylcycloxacarbocyanine; fluorenylmethyloxycarbonyl chloride; 7-amino-4- methylcourmarin (Amc); green fluorescent protein (GFP); and derivatives or combinations thereof.

[0127] In some examples, the detectable moiety can comprise Rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcourmarin (Amc), green fluorescent protein (GFP), naphthofluorescein (NF), or derivatives or combinations thereof. Attorney Docket No. 103362-028WO1

[0128] The detectable moiety can be attached to the cell penetrating peptide moiety at the amino group, the carboxylate group, or the side chain of any of the amino acids of the cell penetrating peptide moiety (e.g., at the amino group, the carboxylate group, or the side chain of any amino acid in the CPP).

[0129] Linker

[0130] In various embodiments, the linker is covalently bound to an amino acid on the membrane translocation domain. The linker may be any moiety which 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 appropriate molecule which is capable of forming two or more bonds with amino acids in the membrane translocation domain and cargo moiety. Thus, in various embodiments, the precursor of the linker has two or more functional groups, each of which are capable of forming a covalent bond to the membrane translocation domain and cargo moiety. For example, the linker can be covalently bound to the N-terminus, C-terminus, or side chain, or combinations thereof, of any amino acid in the membrane translocation domain. In particular embodiments, the linker forms a covalent bond between the membrane translocation domain and cargo moiety. In some embodiments, the linker can be an unstructured polypeptide sequence. In some embodiments, the when the linker is an unstructured polypeptide sequence it allows for the membrane translocation domain, linker, and cargo conjugate to be produced recombinantly.

[0131] In some embodiments, the linker is selected from the group consisting of at least one amino acid, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ether, each of which can be optionally substituted as defined above. Non-limiting examples of linkers include polyethylene glycol, optionally conjugated to a lysine residue.

[0132] In some embodiments, the linker length can be from 0 to 1000 amino acids. In some embodiments, the linker can be designed to be Gly-Gly-Ser repeats. In some embodiments, the linker sequences can be a linker described in Adv Drug Deliv Rev. 2013 October 15; 65(10): 1357-1369. For example, flexible linker sequences defined by (G)n(n=l-10), or SEQ ID NO: 131 (GGGGS)n(n=l-4), such as SEQ ID NO: 131 GGGGS, SEQ ID NO: 132 (GGGGS)s; rigid linker sequences defined by SEQ ID NO: 133 A(EAAAK)nA (n = 2-5), or (XP)n(n = 5-20) wherein X designating any amino acid, preferably Ala, Lys, or Glu such as SEQ ID NO: 134 A(EAAAK)4ALEA(EAAAK)4A, SEQ ID NO: 135 AEAAAKEAAAKA, Attorney Docket No. 103362-028WO1

[0133] SEQ ID NO: 136 PAPAP, (Ala-Pro)n(n = 5-17); cleavable linker sequences such as disulfide, protease sensitive sequences, (e.g., SEQ ID NO: 137 VSQTSKLTRj,AETVFPDVb, SEQ ID NO: 138 PLG^LWA0, SEQ ID NO: 139 RVL^AEA, SEQ ID NO: 140 EDVVC SMSY, SEQ ID NO: 141 GGIEGFQGS", SEQ ID NO: 142 TRHRQPRJ.GWE, SEQ ID NO : 143 AGNRVRRJ.SVG, SEQ ID NO: 144 RRRRRRRj,Rj,Rd, or SEQ ID NO: 145 GFLGj,e, whereaProtease sensitive cleavage sites are indicated with “J,”;bF actor Xla / FVIIa sensitive cleavage;cMatrix metalloprotease- 1 sensitive cleavage sequences, one example provided here;dHIV PR (HIV-1 protease); NS3 protease (HCV protease); Factor Xa sensitive cleavage, respectively; Turin sensitive cleavage; andfCathepsin B sensitive cleavage).

[0134] In some embodiments, the linker is covalently bound to the N or C-terminus of an amino acid on CPP motif, or to a side chain of glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). In particular embodiments, the linker forms a bond with the side chain of glutamine on the CPP motif. In other particular embodiments, the linker described herein has a structure of L-1 or L-2:

[0135] L-1 wherein

[0136] AASis a side chain or terminus of an amino acid on the peptide or staple;

[0137] AACis a 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.

[0138] In some embodiments, the linker is capable of releasing the cargo moiety from the membrane translocation domain after the polypeptide conjugate enters the cytosol of the cell. In some embodiments, the linker contains a group, or forms a group after binding to membrane translocation domain and cargo moiety that is cleaved after cytosolic uptake of the polypeptide conjugate to thereby release the cargo moiety. Non-limiting examples of physiologically cleavable linking group include carbonate, thiocarbonate, thioether, thioester, disulfide, sulfoxide, hydrazine, protease-cleavable dipeptide linker, and the like. Attorney Docket No. 103362-028WO1

[0139] For example, in embodiments, the linker is covalently bound to membrane translocation domain through a disulfide bond e.g., with the side chain of 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 a precursor of the linker, and the side chain of cysteine or an amino acid analog having a thiol group on the peptide, wherein the bond to hydrogen on 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 which can be used with the polypeptide conjugates disclosed herein are discussed above.

[0140] Methods of Making

[0141] The compounds described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

[0142] Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, 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.

[0143] The starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Attorney Docket No. 103362-028WO1

[0144] Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Suppiementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein can be obtained from commercial sources.

[0145] Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, z.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,JH or13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0146] The disclosed compounds can be prepared by expressing and purifying like any other proteins. See Chen, K., & Pei, D. (2020). Engineering Cell-Permeable Proteins through Insertion of Cell -Penetrating Motifs into Surface Loops. ACS chemical biology, 15(9), 2568-2576, which is incorporated by reference herein in its entirety for its teachings of methods of preparing proteins. Other methods for preparing the disclosed compositions involve solid phase peptide synthesis wherein the amino acid a-N-terminal is protected by an acid or base protecting group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein. Suitable protecting groups are 9- fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobomyloxycarbonyl, a,a-dimethyl- 3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. Other preferred side chain protecting groups are, for side chain amino groups like lysine and arginine, 2, 2, 5,7,8- Attorney Docket No. 103362-028WO1 pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene- sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy- carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p- toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for asparticacid and glutamic acid, benzyl and t-butyl and for cysteine, triphenylmethyl (trityl). In the solid phase peptide synthesis method, the a-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are those materials which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the media used. Solid supports for synthesis of a-C- terminal carboxy peptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-l% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin available from Applied Biosystems (Foster City, Calif.). The a-C-terminal amino acid is coupled to the resin by means of N,N'-dicyclohexylcarbodiimide (DCC), N,N'- diisopropylcarbodiimide (DIC) or O-benzotriazol-l-yl-N,N,N',N'- tetramethyluroniumhexafluorophosphate (HBTU), with or without 4- dimethylaminopyridine (DMAP), 1 -hydroxybenzotriazole (HOBT), benzotriazol- 1-yloxy- tris(dimethylamino)phosphoniumhexafluorophosphate (BOP) or bis(2-oxo-3- oxazolidinyl)phosphine chloride (BOPCI), mediated coupling for from about 1 to about 24 hours at a temperature of between 10°C and 50°C in a solvent such as di chloromethane or DMF. When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy- acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, preferably piperidine, prior to coupling with the a-C-terminal amino acid as described above. One method for coupling to the deprotected 4 (2',4'-dimethoxyphenyl-Fmoc- aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-l-yl-N,N,N',N'- tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1 -hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer. In one example, the a-N-terminal in the amino acids of the growing peptide chain are protected with Fmoc. The removal of the Fmoc protecting group from the a-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in about 3-fold molar excess, and the coupling is preferably carried out in DMF. Attorney Docket No. 103362-028WO1

[0147] The coupling agent can be O-benzotriazol-l-yl-N,N,N',N'- tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1 -hydroxybenzotriazole (HOBT, 1 equiv.). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either in successively or in a single operation. Removal of the polypeptide and deprotection can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thianisole, water, ethanedithiol and trifluoroacetic acid. In cases wherein the a-C-terminal 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 by direct transamidation. The protected peptide can be purified at this point or taken to the next step directly. The removal of the side chain protecting groups can be accomplished using the cleavage cocktail described above. The fully deprotected peptide can be purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivitized polystyrene-divinylbenzene (for example, Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g. on Sephadex G-25, LH-20 or countercurrent distribution; high performance liquid chromatography (HPLC), especially reverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phase column packing.

[0148] Methods of Use

[0149] Provided herein are methods of use of the compounds and / or compositions described herein. Also provided are methods of delivering a plant stimulant into a plant cell including contacting the plant cell with the peptide described herein. Also provided are methods of delivering a plant activator into a plant cell including contacting the plant cell with the peptide described herein. Also provided are methods of delivering a plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126 into a plant cell including contacting the plant cell with the peptide described herein.

[0150] Also provided are methods of delivering a plant stimulant into a plant including contacting the plant with the peptide described herein. Also provided are methods of delivering a plant activator into a plant including contacting the plant with the peptide described herein. Also provided are methods of delivering a plant bioactive moiety Attorney Docket No. 103362-028WO1 including SEQ ID NO: 126 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126 into a plant including contacting the plant with the peptide described herein.

[0151] Also described are methods of treating a plant that has a disease caused by a pathogenic agent. The method can include contacting a plant with an effective amount of a compound and / or composition described herein.

[0152] In some aspects, the compounds or compositions described herein can be used to protect plants against biotic stress caused by living organisms, such as fungi, bacteria, nematodes, insects, mites, and animals; stimulate seeds during germination; to protect plants against abiotic stress caused by a physical or chemical stressor of non-living origin such as the presence of harmful chemicals including salts, restricted access to water, sunscald, freeze injury, wind injury, nutrient deficiency, or improper cultural practices, such as overwatering or planting too deep; to enhance growth, yield, health, longevity, productivity, and / or vigor of a plant; and / or provide multiple disease resistance to a plant.

[0153] Provided are also methods to protect plants against biotic stress; stimulate seeds during germination; to protect plants against abiotic stress; to enhance growth, yield, health, longevity, productivity, and / or vigor of a plant; to provide multiple disease resistance to a plant; or any combination thereof. The methods can include contacting the plant cell with the peptide described herein.

[0154] The compounds or compositions described herein provides plants’ resistance to a diverse range of pathogens. In some aspects, the compounds or compositions can be used as a plant stimulant for plants that have a disease caused by a pathogenic agent. The pathogenic agent can include a fungus, virus, bacterium, mycoplasm, spiroplams or viroid. Exemplary pathogens may include fungi, such as Erisyphe polygoni, Phytophthora capsicci. Verticillium dahliae and other Verticillium spp., Powdery mildew, and Fusarium spp. bacteria, such as Pseudomonas syringae py. tomato (e.g. Pst DC3000), and viruses, such as tobacco mosaic virus and brome mosaic virus. Other exemplary pathogens include Colletotrichum lagenarum. Pyricularia oryzae, Pseudomonas lachrymans. Xanthomonas oryzae, Xanthomonas euvesicatoria, Xanthomonas gardneri. Xanthomonas perforans, and Xanthomonas vesicatoria, Phytophthora infestants on tomatoes, Plasmopara viticola, Pseudomonas tomato, Phytophthora parasitica var. nicoliniae, Peronospora lahacina, Cercospora nicolianae. Pseudomonas lahaci, Erysiphe graminis, Phytophora medicaginis, P. megasperma, Pyricularia oryzae, Helminthosporium leaf blight such as Attorney Docket No. 103362-028WO1

[0155] Helminthosporium oryzae, Cochliobolus miyabeanus, Bakanae disease such as Gibberella fiijikiiroi. seedling blight such as Rhizopus oryzae, sheath blight such as Rhizoctonia solani, Puccinia coronala, powdery mildew such as 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, Alternaria leaf spot , Liberibacter spp. (L. asiaticus, L. africanus, L. americanus) that cause citrus greening disease or Huanglongbing. Also the soybean rust pathogen: Phakopsora pachyrhizi and other diseases of grains, cereals, beet, leguminous plants, pomes, drupes, fruits, citrus fruit, oil plants, cucumber plants, fiber plants, lauraceae, ornamentals, and vegetables such as oil-seed rape, sunflower, carrot, pepper, strawberry, melon, kiwi fruit, onion, leek, sweet potato, fig, ume, asparagus, persimmon, soybean, adzuki-bean, watermelon, crown daisy, spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, paprika, tea, wheat, barley, rye, oats, rice, sorghum, sugar beet, fodder beet, apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries, beans, lentils, peas, soybeans, rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts, cucumber, marrows, melons, cotton, flax, hemp, jute, oranges, lemons, grapefruit, mandarins, avocados, cinnamon, camphor, maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, bananas, natural rubber plants, flowers, shrubs, deciduous trees and conifers, and such the like.

[0156] The compounds or compositions described herein can be effective against a wide variety of insects. European corn borer is a major pest of corn (dent and sweet com) but also feeds on over 200 plant species, including green beans, wax beans, lima beans, soybeans, peppers, potato, tomato, and many weed species. Additional insect larval feeding pests which damage a wide variety of vegetable crops include, without limitation, beet armyworm, cabbage looper, com ear worm, fall armyworm, diamondback moth, cabbage root maggot, onion maggot, seed corn maggot, pickleworm (melonworm), pepper maggot, and tomato pinworm. Attorney Docket No. 103362-028WO1

[0157] With regard to the use of the 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 begins from seeds or later in the life of a plant. For example, plant growth according to the present invention encompasses greater yield, increased quantity of seeds produced, increased percentage of seeds germinated, increased plant size, greater biomass, more and bigger fruit, earlier fruit coloration, and earlier fruit and plant maturation. For example, early germination and early maturation permit crops to be grown in areas where short growing seasons would otherwise preclude their growth in that locale. Increased percentage of seed germination results in improved crop stands and more efficient seed use. Greater yield, increased size, and enhanced biomass production allow greater revenue generation from a given plot of land. In some embodiments, the compounds or compositions can be used to promote early flowering.

[0158] As used herein, “health of a plant” or “plant health” means the condition of a plant and / or its products which is determined by several aspects alone or in combination with each other, such as increased yield, plant vigor, quality, and tolerance to abiotic and / or biotic stress.

[0159] A plant suffering from pathogens or insecticidal attack often produces a smaller biomass, which leads to a reduced yield as compared to a plant which has been subjected to curative or preventive treatment against the pathogenic fungus or any other relevant pest and which can grow without the damage caused by the biotic stress factor. However, applying a compound and / or composition described herein leads to enhanced plant health even in the absence of any biotic stress. The application of the compound and / or composition described herein to a plant and / or area of cultivation can also be carried out in the absence of pest pressure on the plant.

[0160] According to the present invention, “increasing 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 without application of the compound and / or composition to the plant and / or area of cultivation. In one embodiment, the term “yield” refers to fruits in the proper sense, as well as vegetables, nuts, grains, and seeds.

[0161] “Grain” and “fruit” are to be understood as any plant product which is further utilized after harvesting, e.g., fruits in the proper sense, vegetables, nuts, grains, seeds, Attorney Docket No. 103362-028WO1 wood (e.g., in the case of silviculture plants), flowers (e.g., in the case of gardening plants and ornamentals), etc.

[0162] Increased yield of a plant can be characterized by the following non-limiting properties: increased plant weight; increased biomass, such as higher overall fresh weight (FW) and / or higher overall dry weight (DW); increased number of flowers per plant; higher grain and / or fruit yield; more tillers or side shoots (branches); larger leaves; increased shoot growth; increased protein content; increased oil content; increased starch content; increased pigment content; increased chlorophyll content; and any combination thereof.

[0163] Chlorophyll content has a positive correlation with a plant's photosynthesis rate and, accordingly, the higher the chlorophyll content the higher the yield of a plant.

[0164] Increasing the yield of a plant may involve improving plant vigor. Plant vigor becomes manifest in several aspects, including the general visual appearance of the plant. Improved plant vigor can be characterized by, inter alia, the following: improved vitality of the plant; improved plant growth; improved plant development; improved visual appearance; improved plant stand (less plant verse / lodging); improved emergence; enhanced root growth and / or more developed root system; enhanced nodulation, in particular rhizobial nodulation; bigger leaf blade; bigger size; increased plant height; increased tiller number; increased number of side shoots; increased number of flowers per plant; increased shoot growth; increased root growth (extensive root system); enhanced photosynthetic activity; enhanced pigment content; earlier flowering; earlier fruiting; earlier and improved germination; earlier grain maturity; fewer non-productive tillers; fewer dead basal leaves; less input needed (such as fertilizers or water); greener leaves; complete maturation under shortened vegetation periods; less fertilizer needed; fewer sowing of seeds needed; easier harvesting; faster and more uniform ripening; longer shelf-life; longer panicles; delay of senescence; stronger and / or more productive tillers; better extractability of ingredients; improved quality of seeds (for being seeded in the following seasons for seed production); reduced production of ethylene and / or the inhibition of its reception by the plant; and any combination thereof.

[0165] Enhanced photosynthetic activity of a plant may be based on increased stomatai conductance and / or an increased CO2 assimilation rate of the plant.

[0166] Increasing the yield of a plant may involve improving the quality of a plant and / or its products. Improvements in plant quality may include, without limitation, improving certain plant characteristics, such as increasing the content and / or composition of certain Attorney Docket No. 103362-028WO1 ingredients by a measurable or noticeable amount over the same factor of the plant produced under the same conditions, but without application of the composition of the present invention. Enhanced quality can be characterized by, inter alia, the following: increased nutrient content; increased protein content; increased content of fatty acids; increased metabolite content; increased carotenoid content; increased sugar content; increased amount of essential amino acids; improved nutrient composition; improved protein composition; improved composition of fatty acids; improved metabolite composition; improved carotenoid composition; improved sugar composition; improved amino acids composition; improved or optimal fruit color; improved leaf color; higher storage capacity; higher processability of the harvested products; or any combination thereof.

[0167] Increasing the yield of a plant may involve improving a plant's tolerance or resistance to biotic and / or abiotic stress factors. Biotic and abiotic stress, especially over longer terms, can have harmful effects on plants. Biotic stress is caused by living organisms while abiotic stress is caused, for example, by environmental extremes. In one embodiment, applying the compound and / or composition described herein to a plant pursuant to the method of the present invention enhances tolerance or resistance to biotic and / or abiotic stress factors, meaning: (1) certain negative factors caused by biotic and / or abiotic stress are diminished in a measurable or noticeable amount as compared to plants exposed to the same conditions, but without being treated with a compound and / or composition described herein and (2) the negative factors are not diminished by a direct action of the composition on the stress factors, e.g., by its fungicidal or insecticidal action which directly destroys the microorganisms or pests, but rather by a stimulation of the plants' own defensive reactions against said stress factors.

[0168] Negative factors caused by biotic stress, such as pathogens and pests, are widely known and range from dotted leaves to total destruction of the plant. Biotic stress can be caused by living organisms, such as pests (e.g., insects, arachnides, and nematodes), competing plants (e.g., weeds), microorganisms (e.g., phytopathogenic fungi and / or bacteria), and / or viruses.

[0169] Negative factors caused by abiotic stress are also well-known and can often be observed either as reduced plant vigor (as described above) or by the following symptoms: dotted leaves, “burned” leaves, reduced growth, fewer flowers, less biomass, less crop yield, reduced nutritional value of the crop, and later crop maturity, to give just a few examples. Attorney Docket No. 103362-028WO1

[0170] Abiotic stress can be caused by, inter alia: extremes in temperature such as heat or cold (heat stress / cold stress), strong variations in temperature, temperatures unusual for the specific season, drought (drought stress), extreme wetness, high salinity (salt stress), radiation (e.g., by increased UV radiation due to the decreasing ozone layer), increased ozone levels (ozone stress), organic pollution (e.g., by phytotoxic amounts of pesticides), inorganic pollution (e.g., by heavy metal contaminants), and any combination thereof.

[0171] Biotic and / or abiotic stress factors decrease the quantity and the quality of the stressed plants, their crops, and fruits. As far as quality is concerned, reproductive development can be affected with consequences on the crops which are important for fruits or seeds. Synthesis, accumulation, and storage of proteins are mostly affected by temperature; growth is slowed by almost all types of stress; polysaccharide synthesis, both structural and storage, is reduced or modified. These effects result in a decrease in biomass (yield) and in changes in the nutritional value of the plant product.

[0172] The above identified indicators for the health condition of a plant may be interdependent and may result from each other. For example, an increased resistance to biotic and / or abiotic stress may lead to a better plant vigor, e.g., to better and bigger crops, and thus to an increased yield. Inversely, a more developed root system may result in an increased resistance to biotic and / or abiotic stress.

[0173] Applying the compound and / or composition described herein to a plant and / or area of cultivation can have a synergistic effect on the plant to: increase the health of the plant, increase the yield of the plant, increase the biomass of the plant, increase the oil content of the plant, increase the vigor of the plant, increase the stand of the plant, increase the emergence of the plant, increase the root growth of the plant, increase the photosynthetic activity of the plant, improve the quality of the plant, improve the nutrient composition of the plant, improve the protein composition of the plant, improve the carotenoid composition of the plant, increase the tolerance of the plant to biotic stress, increase the tolerance of the plant to fungi, increase the tolerance of the plant to nematodes, increase the tolerance of the plant to bacteria, increase the tolerance of the plant to abiotic stress, increase the tolerance of the plant to drought stress, increase the tolerance of the plant to cold stress, increase the tolerance of the plant to heat stress, increase the tolerance of the plant to salt stress, increase the tolerance of the plant to ozone stress, and / or any combination thereof.

[0174] One of the most important factors for increased resistance against biotic and abiotic stress is the stimulation of the plant's natural defense reactions, which occurs by application Attorney Docket No. 103362-028WO1 of compound and / or composition described herein according to the method described herein.

[0175] The plant, including its roots, flowers, leaves, or stems, can be contacted with the disclosed compounds or compositions in any known technique for applying plant formulations.

[0176] Exemplary application techniques include, but are not limited to, spraying, atomizing, dusting, spreading, sprinkling, dripping, dipping, drenching, injecting, hydrophonics, or direct application into water (in-water). In some embodiments, suitable compositions can include those for HV, LV, and ULV spraying and for ULV cool and warm fogging formulations. The method of application can vary depending on the intended purpose. The compositions can be applied on the plants in a field or in a greenhouse. In some aspects, the compositions can be applied to a portion of the plant, for example, to the tubers before planting. In some embodiments, the compounds or compositions can be applied on the plants surface or plant plasma membrane as a foliar spray.

[0177] The composition can be contacted with any part of the plant, for example, the root or the leaves of the plant. In some embodiments, the composition can be contacted to the roots by spraying the soil, mechanical incorporation, mixed with fertilizer, soil improvement, premix or such the like.

[0178] In some embodiments, the composition can be contacted to a plant seed. Seed treatments containing the compounds or compositions can be applied using any commercially available seed treatment machinery or can also be applied using any acceptable non-commercial method(s) such as the use of syringes or any other seed treatment device. General seed treatments coating procedures using compounds or compositions can be performed using a Wintersteiger HEGE 11 (Wintersteiger AG, Austria, Germany) and applied to the seed of major crops, namely com, soybean, wheat, rice and various vegetables.

[0179] The seeds can be coated using a variety of methods including, but not limited to, pouring or pumping, drizzling or spraying an aqueous solution containing the compounds or compositions on or over a seed, spraying or applying onto a layer of seeds either with the use or without the use of a conveyor system. Suitable mixing devices include tumblers, mixing basins or drums, or other fluid applicating devices that include basins or drums used to contain the seed while coating. Attorney Docket No. 103362-028WO1

[0180] The compositions described herein can be contacted intermittently to the plant. In some aspects, the plant can be contacted with the composition two times of greater. For example, the plant can be contacted with the composition 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the plant can be contacted with the composition from 2 to about 5 times. In some embodiments, the plant can be contacted with the composition once. In some aspects, the plant can be contacted with the composition once every 5 to 21 days. For example, the plant can be contacted with the composition once every 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the plant can be contacted with the composition once per week. In some aspects, the plant can be contacted with the composition 1 to 5 times per 5 to 21 days. For example, the plant can be contacted about 1 to about 5 times per week.

[0181] In some aspects the compositions described herein can be applied before the stressing factor(s) appears.

[0182] The term “plant” as used herein includes whole plants and parts thereof, including, but not limited to, shoot vegetative organs / structures (e.g., leaves, stems and tubers), roots, flowers and floral organs / structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, and the like), and progeny of same. A “plant cell” as used herein refers to any plant cell and can comprise a cell at the plant surface or internal to the plant plasma membrane, for example, an epidermal cell, a trichome cell, a xylem cell, a phloem cell, a sieve tube element, or a companion cell.

[0183] The class of plants that can be used in the methods described herein include the class of higher and lower plants, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae. For example, plants for use in the methods described herein include any vascular plant, for example monocotyledons or dicotyledons or gymnosperms, including, but not limited to alfalfa, apple, Arabidopsis, banana, barley, canola, castor bean, chrysanthemum, clover, cocoa, coffee, cotton, cottonseed, corn, crambe, cranberry, cucumber, dendrobium, dio-scorea, eucalyptus, fescue, flax, gladiolus, liliacea, linseed, millet, muskmelon, mustard, oat, oil palm, oilseed rape, papaya, peanut, pineapple, ornamental plants, Phaseolus, potato, rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, sugarbeet, sugarcane, sunflower, strawberry, tobacco, tomato, turfgrass, Attorney Docket No. 103362-028WO1 wheat and vegetable crops such as lettuce, celery, broccoli, cauliflower, cucurbits, onions (including garlic, shallots, leeks, and chives); fruit and nut trees, such as apple, pear, peach, orange, grapefruit, lemon, lime, almond, pecan, walnut, hazel; vines, such as grapes, kiwi, hops; fruit shrubs and brambles, such as raspberry, blackberry, gooseberry; forest trees, such as ash, pine, fir, maple, oak, chestnut, popular; with alfalfa, canola, castor bean, corn, cotton, crambe, flax, linseed, mustard, oil palm, oilseed rape, peanut, potato, rice, safflower, sesame, soybean, sugarbeet, sunflower, tobacco, tomato, and wheat preferred. In some embodiments, plants for use in the methods described herein include any crop plant, for example, forage crop, oilseed crop, grain crop, fruit crop, vegetable crop, fiber crop, spice crop, nut crop, turf crop, sugar crop, beverage crop, and forest crop.

[0184] Plants may be categorized into agricultural, silvicultural, ornamental, and horticultural plants, based on their human use and / or consumption. In addition, “plants” include natural or wildtype plants, and plants that have been genetically modified.

[0185] “Agricultural” plants are plants of which a part or all is harvested or cultivated on a commercial scale or which serve as an important source of feed, food, fibers (e.g., cotton and linen), combustibles (e.g., wood, bioethanol, biodiesel, and biomass) or other chemical compounds. Agricultural plants also include vegetables. Thus, agricultural plants include cereals (e.g., wheat, rye, barley, triticale, oats, sorghum, and rice); beet (e.g., sugar beet or fodder beet); leguminous plants (e.g., beans, lentils, peas, alfalfa, and soybean); oil plants (e.g., rape, oil-seed rape, canola, juncea (Brassica juncea). linseed, mustard, olive, sunflower, cocoa bean, castor oil plants, oil palms, ground nuts, and soybean); cucurbits (e.g., squash, cucumber, and melon); fiber plants (e.g., cotton, flax, hemp, and jute); vegetables (e.g., cucumbers, spinach, lettuce, asparagus, cabbages, carrots, radish, turnip, celery, chicory, endive, brussel sprouts, parsnip, cauliflower, broccoli, garlic, eggplant, pepper, pumpkin, onions, tomatoes, potatoes, sweet potatoes, cucurbits, and paprika); lauraceous plants (e.g., avocados, cinnamon, and camphor); energy and raw material plants (e.g., corn, soybean, rape, canola, sugar cane, and oil palm); tobacco; nuts (including peanuts); coffee; tea; vines (e.g., table grapes and juice grape vines); hop; stone fruit; apple; blueberry; strawberry; pear; citrus; raspberry; pineapple; sugarcane; turf, natural rubber plants, and marijuana.

[0186] “Horticultural plants” are plants commonly used in horticulture and include, without limitation, ornamentals, vegetables, and fruits. “Ornamental” plants are plants which are commonly used in gardening, e.g., in parks, gardens, and on balconies and patios. Non- Attorney Docket No. 103362-028WO1 limiting examples of ornamentals include turf, geranium, pelargonia, petunia, begonia, and fuchsia. Non-limiting examples of vegetables are as described above. Non-limiting examples of fruits include apples, pears, cherries, strawberry, citrus, peaches, apricots, and blueberries.

[0187] “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 wood, pulp, paper, rubber tree, Christmas trees, or young trees for gardening purposes. Non-limiting examples of silvicultural plants are conifers (e.g., pines), in particular Pinus species fir and spruce; eucalyptus,' tropical trees (e.g., teak, rubber tree, oil palm); willow (Salix), in particular Salix species; poplar (cottonwood), in particular Populus species; beech, in particular Fagus species; birch; oil palm; cherry, walnut, and oak.

[0188] As noted above, the term “plant” also includes plants modified from their wildtype form. Such modifications may occur through breeding, mutagenesis, or genetic engineering (including transgenic and non-transgenic plants). Plants modified by genetic engineering include plants having genetic material that has been modified by the use of recombinant DNA techniques. Such modifications typically include modifications that cannot readily be obtained by cross breeding under natural circumstances, mutations, or natural recombination. Typically, one or more genes have been integrated into the genetic material of a genetically modified plant in order to improve certain properties of the plant. Examples of genetically modified plants, include but are not limited to, crops which tolerate the action of herbicides, fungicides, or insecticides owing to breeding, including genetic engineering methods, or plants which have modified characteristics in comparison with existing plants, which can be generated by, e.g., traditional breeding methods and / or the generation of mutants, or by recombinant procedures. Examples of genetically modified plants also include those that, through the use of recombinant DNA techniques, are able to synthesize one or more proteins to increase the resistance or tolerance of those plants to bacterial, viral, or fungal pathogens; to increase the productivity (e.g., biomass production, grain yield, starch content, oil content, and / or protein content), tolerance to drought, salinity, or other growth-limiting environmental factors or tolerance to pests and fungal, bacterial, or viral pathogens of those plants, and / or that contain a modified amount of substances of content or new substances of content, specifically to improve raw material production and / or to improve human or animal nutrition, e.g., potatoes that produce increased amounts of Attorney Docket No. 103362-028WO1 amylopectin (e.g., oil crops that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids (e.g., Nexera® rape, DOW Agro Sciences, Canada), Amflora® potato, BASF SE, Germany).

[0189] The methods for producing such genetically modified plants are generally known to the person of ordinary skill in the art and are described, e.g., in the above-noted references.

[0190] Depending on the intended mode of administration, the compositions described herein can be in the form of a solid, a semi-solid, a liquid, a solution, a suspension, an emulsion, a gel, an oil dispersion, capsule (such as the active ingredient encapsulated in a microcapsule), or the like. In some embodiments, the compositions can include, as noted above, an agriculturally effective amount of the compound described herein in combination with an agriculturally acceptable carrier and, in addition, can include other carriers, adjuvants, diluents, thickeners, buffers, preservatives, surfactants, etc. In some aspects, concentrates, suitable for dilution, of the compositions can be prepared with the compositions, in addition to water, a wetting agent, a tackifier, a dispersant, or an emulsifier.

[0191] The agriculturally acceptable carrier can include an organic or an inorganic carrier. Exemplary carriers include, but are not limited to, water, organic solvents, inorganic solvents, petroleum fractions or hydrocarbons such as mineral oil, aromatic solvents, paraffinic oils, vegetable oils such as soybean oil, rapeseed oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil, esters of the above vegetable oils, esters of monoalcohols or dihydric, trihydric, or other lower polyalcohols (4-6 hydroxy containing), such as 2-ethyl hexyl stearate, n-butyl oleate, isopropyl myristate, propylene glycol dioleate, di-octyl succinate, di-butyl adipate, di-octyl phthalate, esters of mono, di and polycarboxylic acids, toluene, xylene, petroleum naphtha, crop oil, 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, glycerine, N- methyl-2-pyrrolidinone, M-V-di methyl alkylamides, dimethyl sulfoxide, liquid fertilizers, and mixtures thereof. Other exemplary carriers include silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, pyrophyllite clay, attapulgus clay, kieselguhr, calcium carbonate, bentonite clay, Fuller's earth, cottonseed Attorney Docket No. 103362-028WO1 hulls, wheat flour, soybean flour, pumice, wood flour, walnut shell flour, lignin, ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas, cereal meal, tree bark meal, wood meal and nutshell meal, cellulose powders, and mixtures thereof. The agriculturally acceptable carrier can 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.

[0192] Exemplary agriculturally acceptable adjuvants include, but are not limited to, antifreeze agents, antifoam agents, compatibilizing agents, sequestering agents, neutralizing agents and buffers, corrosion inhibitors, colorants, odorants, penetration aids, wetting agents, spreading agents, dispersing agents, thickening agents, freeze point depressants, antimicrobial agents, crop oil, safeners, adhesives, surfactants, protective colloids, emulsifiers, tackifiers, and mixtures thereof. The agriculturally acceptable adjuvant can be present in an amount of 15% by volume or less, 10% by volume or less, or 5% by volume or less, based on the volume of the composition.

[0193] The compositions described herein can be in any suitable form based on its intended use. In some aspects, the compositions can be in the form of an aqueous solution. In some aspects, the compositions can be a solution comprising an organic solvent, such as an alcohol. In some aspects, the compositions can be a solution comprising a mixture of organic and inorganic solvents. In some aspects, the compositions can be in the form of an emulsion.

[0194] The compositions described herein can include an additional plant protection composition. For example, the compositions can include a fungicidal agent, an antiviral agent, an antibacterial agent, or a combination thereof.

[0195] The compositions described herein can comprise from 0.001 to 99% by weight of active compound, that is the compound described herein, together with the carriers and / or adjuvants.

[0196] Methods of using the compositions as a plant stimulant are also described herein. The method can include contacting a plant with an effective amount of a composition comprising a compound described herein. The plant, including its roots, flowers, leaves, or stems, can be contacted with the disclosed compounds or compositions in any known Attorney Docket No. 103362-028WO1 technique for applying plant stimulants. Exemplary application techniques include, but are not limited to, spraying, atomizing, dusting, spreading, sprinkling, dripping, dipping, drenching, injecting, hydrophonics, or direct application into water (in-water). The method of application can vary depending on the intended purpose. The compositions can be applied on the plants in a field or in a greenhouse. In some aspects, the compositions can be applied to a portion of the plant, for example, to the tubers before planting.

[0197] The composition can be contacted with any part of the plant, for example, the root or the leaves of the plant. In some embodiments, the composition can be contacted to the roots by spraying the soil, mechanical incorporation, mixed with fertilizer, soil improvement, premix or such the like.

[0198] The selected dosage level of the composition will depend upon a variety of factors including for example, the activity of the compound according to Formula I, the route of administration, the time of administration, the duration of the treatment, other drugs and / or materials used in combination with the particular compound employed, the condition and general health of the plant being treated, and like factors well-known in the agricultural arts. However, the compositions described herein provides plant stimulation even at low doses. In some embodiments, wherein the compositions disclosed herein are less well tolerated by certain crop plants, the compositions can be applied with the aid of the spray apparatus in such a way that they come into little contact, if any, with the leaves of the sensitive crop plants while reaching the leaves of undesirable vegetation that grows underneath or the bare soil (e.g., post-directed or lay-by). A person having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required.

[0199] The compositions described herein can be contacted intermittently to the plant. In some aspects, the plant can be contacted with the composition two times of greater. For example, the plant can be contacted with the composition 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the plant can be contacted with the composition from 2 to about 5 times. In some embodiments, the plant can be contacted with the composition once. In some aspects, the plant can be contacted with the composition once every 5 to 21 days. For example, the plant can be contacted with the composition once every 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the plant can be contacted with the composition once per week. In some aspects, the plant can be contacted with the composition 1 to 5 times per 5 to 21 days. For example, the plant can be contacted about 1 to about 5 times per week. Attorney Docket No. 103362-028WO1

[0200] In some aspects the compositions described herein can be applied before the stressing factor(s) appears.

[0201] The compositions can used in combination with an additional plant protection product. For example, the composition can be used with a fungicidal agent, an antiviral agent, or an antibacterial agent. In some aspects, the method of stimulating a plant can include applying an additional plant protection product and a composition comprising a compound described herein.

[0202] The composition including a compound described herein and the fungicidal agent, antiviral agent, or antibacterial agent can be applied to the plant simultaneously or sequentially. In some embodiments, the fungicidal agent, antiviral agent, or antibacterial agent is applied to the plant after the composition comprising a compound described herein.

[0203] In some aspect, the fungicidal agent, antiviral agent, or antibacterial agent and the compound described herein are applied in a synergistically effective amount. As described in the Herbicide Handbook of the Weed Science Society of America, Ninth Edition, 2007, p. 429, “‘synergism’ [is] an interaction of two or more factors such that the effect when combined is greater than the predicted effect based on the response to each factor applied separately.” Synergistic in the herbicide context can mean that the use of the fungicidal agent, antiviral agent, or antibacterial agent and the compound described herein results in an increased stimulating effect compared to the stimulating effects that are possible with the use of each compound alone. In some embodiments, the fungicidal agent, antiviral agent, or antibacterial agent is applied at a rate of 50% or less the recommended rate. For example, the fungicidal agent, antiviral agent, or antibacterial agent is applied at a rate of 45% or less, 40% or less, 35% or less, or 33% or less the recommended rate.

[0204] Compositions, Formulations and Methods of Administration

[0205] Also disclosed herein are compositions comprising the compounds described herein. Agricultural formulations of active substances are well known. Non-limiting examples include a solid, a semi-solid, a liquid, a solution, a suspension, an emulsion, a gel, an oil dispersion, capsule (such as the active ingredient encapsulated in a microcapsule), dusts, powders, pastes, granules, or the like. The particular formulation chosen may vary depending on the particular intended mode of administration. In each case, it is typically an advantage to ensure a fine and even distribution of the active ingredient(s) in a liquid or solid carrier. The compositions described herein can be in any suitable form based on its intended use. In some aspects, the compositions can be in the Attorney Docket No. 103362-028WO1 form of an aqueous solution. In some aspects, the compositions can be a solution comprising an organic solvent, such as an alcohol. In some aspects, the compositions can be a solution comprising a mixture of organic and inorganic solvents.

[0206] Preferably, the composition described herein is formulated in a manner suitable for large or small scale agricultural and horticultural applications.

[0207] The selected dosage level of the composition will depend upon a variety of factors including for example, the route of administration, the time of administration, the duration of the treatment, other drugs and / or materials used in combination with the particular compound employed, the condition and general health of the plant being treated, and like factors well-known in the agricultural arts. However, the compositions described herein provides plant stimulation even at low doses. A person having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required.

[0208] Formulation methods are taught, e.g., 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 E.I. 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.; U.S. Pat. No. 5,208,030 to Hoy et al., Great Britain Patent No. 2,095,558; U.S. Pat. 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 hereby incorporated by reference in its entirety.

[0209] The compositions can include, as noted above, an agriculturally effective amount of the compound described herein in combination with an agriculturally acceptable carrier and, in addition, can include other auxiliaries. The compositions can be formulated (either together or separately) in a manner common for agrochemical formulations. For example, the composition(s) may include auxiliaries which are customary in agrochemical formulations. The particular auxiliaries used may depend on the particular application form and active substance, respectively. Non-limiting examples of suitable auxiliaries include carriers, adjuvents, diluents, thickeners, buffers, preservatives, surfactants, wetting agent, a coating agent, a monosaccharide, a polysaccharide, an abrading agent, a pesticide, an Attorney Docket No. 103362-028WO1 insecticide, an herbicide, a nematicide, a bacteriocide, a fungicide, a miticide, a fertilizer, a biostimulant, a colorant, a humectant, antifreeze agents, antifoam agents, compatibilizing agents, sequestering agents, neutralizing agents and buffers, corrosion inhibitors, an osmoprotectant, odorants, an antibiotic, spreading agents, dispersing agents, freeze point depressants, antimicrobial, crop oil, safeners, adhesives, protective colloids, emulsifiers, tackifiers, an amino acid, a biological control agent, or a combination thereof.

[0210] In some embodiments, the compositions described herein can include a pesticide comprising an insecticide, a herbicide, a fungicide, a bacteriocide, a nematicide, a miticide, or any combination thereof. The pesticide can be applied to the plant simultaneously or sequentially. In some embodiments, pesticide is applied to the plant after the compound and / or composition described herein is applied.

[0211] In some aspects, concentrates, suitable for dilution, of the compositions can be prepared with the compositions, in addition to water, a wetting agent, a tackifier, a dispersant, or an emulsifier.

[0212] The agriculturally acceptable carrier can include an organic or an inorganic carrier. Exemplary carriers include, but are not limited to, water, organic solvents, inorganic solvents, petroleum fractions or hydrocarbons such as mineral oil, aromatic solvents, paraffinic oils, vegetable oils such as soybean oil, rapeseed oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil, esters of the above vegetable oils, esters of monoalcohols or dihydric, trihydric, or other lower polyalcohols (4-6 hydroxy containing), such as 2-ethyl hexyl stearate, n-butyl oleate, isopropyl myristate, propylene glycol dioleate, di-octyl succinate, di-butyl adipate, di-octyl phthalate, esters of mono, di and polycarboxylic acids, toluene, xylene, petroleum naphtha, crop oil, 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, glycerine, N-methyl-2-pyrrolidinone, M -di methyl alkylamides, dimethyl sulfoxide, liquid fertilizers, and mixtures thereof. Other exemplary carriers include silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, pyrophyllite clay, attapulgus clay, kieselguhr, calcium carbonate, bentonite clay, Fuller's earth, cottonseed hulls, wheat Attorney Docket No. 103362-028WO1 flour, soybean flour, pumice, wood flour, walnut shell flour, lignin, ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas, cereal meal, tree bark meal, wood meal and nutshell meal, cellulose powders, and mixtures thereof. The agriculturally acceptable carrier can 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.

[0213] When the composition includes an amino acid, the amino acid can be provided separately from the amino acids that comprise the polypeptide. For example, an isolated amino acid can be used. Suitable amino acids include any natural or unnatural amino acids. For example, the composition can comprise cysteine.

[0214] Unless otherwise specified, each agriculturally acceptable auxiliary can be present from 0.1 to 60 wt. %, from 0.5 to 50 wt. %, or from 10 to 30 wt. % of the total weight of the composition.

[0215] When the composition includes a preservative, the preservative can comprise those based on dichlorophene and benzylalcohol hemi formal (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). As further examples, suitable preservatives include MIT (2-methyl-4- isothiazolin-3-one), BIT (l,2-benzisothiazolin-3-one, which can be obtained from Avecia, Inc. as PROXEL GXL as a solution in sodium hydroxide and dipropylene glycol), 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 hem iformal, or any combination thereof.

[0216] When the composition includes a buffering agent, the buffering agent can comprise potassium, phosphoric acid, a phosphate salt, citric acid, a citrate salt, a sulfate salt, MOPS, or HEPES. The buffering agent can stabilize the polypeptide in the composition. Attorney Docket No. 103362-028WO1

[0217] When the composition includes a wetting agent, the wetting agent can comprise organosilicones, polyoxyethoxylates, polysorbates, polyethyleneglycol and derivatives thereof, ethoxylates, crop oils, and polysaccharides.

[0218] When the composition includes a surfactant, the surfactant can comprise a heavy petroleum oil, a heavy petroleum distillate, a polyol fatty acid ester, a polyethoxylated fatty acid ester, an aryl alkyl polyoxyethylene glycol, a polyoxyethylenepolyoxypropylene monobutyl ether, an alkyl amine acetate, an alkyl aryl sulfonate, a polyhydric alcohol, an alkyl phosphate, an alcohol ethoxylate, an alkylphenol ethoxylate, an alkyphenol ethoxylate, an alkoxylated polyol, an alky polyethoxy ether, an alkylpolyoxethylene glycerol, ethoxylated and soybean oil derivatives, an organosilicone-based surfactant or any combination thereof. Surfactants can be included in a range of compositions including those for foliar use.

[0219] When the composition includes a coating agent, the coating agent can comprise a tackifier, polymers, filling agents, or bulking agents.

[0220] The tackifier can include, but is not limited to, carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules, or latexes, such as gum Arabic, chitin, polyvinyl alcohol and polyvinyl acetate, as well as natural phospholipids, such as cephalins and lecithins, and synthetic phospholipids. Tackifiers include those composed preferably of an adhesive polymer that can be natural or synthetic without phytotoxic effect on the seed to be coated. Additional tackifiers that can be included, either alone or in combination, include, for example, polyesters, polyether esters, polyanhydrides, polyester urethanes, polyester amides; polyvinyl acetates; polyvinyl acetate copolymers; polyvinyl alcohols and tylose; polyvinyl alcohol copolymers; polyvinylpyrolidones; polysaccharides, including starches, modified starches and starch derivatives, dextrins, maltodextrins, alginates, chitosanes and celluloses, cellulose esters, cellulose ethers and cellulose ether esters including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; fats; oils; proteins, including casein, gelatin and zeins; gum arabics; shellacs; vinylidene chloride and vinylidene chloride copolymers; lignosulfonates, in particular calcium lignosulfonates; polyacrylates, polymethacrylates and acrylic copolymers; polyvinylacrylates; polyethylene oxide; polybutenes, polyisobutenes, polystyrene, polybutadiene, polyethyleneamines, polyethylenam ides; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, Attorney Docket No. 103362-028WO1 methylacrylamide monomers; and polychloroprene, or any combination thereof. Tackifiers can be used in a range of compositions including those for seed treatment.

[0221] When the composition includes an abrading agent, the abrading agent can comprise talc, graphite, or a combination of both.

[0222] A humectant is a hygroscopic substance that assists with the retention of moisture. When the composition includes a humectant, the humectant can comprise: glycerol, glycerin, a glycerol derivative (e.g. glycerol monosterate, glycerol triacetate, triacetin, propylene glycol, hexylene glycol, or butylene glycol), triethylene glycol, tripolypropylene glycol, glyceryl triacetate, sucrose, tagatose, a sugar alcohol or a sugar polyol (e.g glycerol, sorbitol, xylitol, mannitol, or mantitol), a polymeric polyol (e.g. polydextrose, a collagen, an aloe or an aloe vera gel), or an alpha hydroxy acid (e.g. lactic acid, honey, molasses, quillaia, sodium hexametaphosphate, lithium chloride or urea). Synthetic humectants can also comprise: butylene glycol, and tremella extract.

[0223] When the compound described herein are formulated or applied in combination with commercially available fungicides, the compositions can provide an extra layer of protection for enhancing disease prevention or spread in a plant.

[0224] A variety of colorants may be employed, including organic chromophores classified as nitroso, nitro, azo, including monoazo, bisazo, and poly azo, diphenylmethane, triarylmethane, xanthene, methane, acridine, thiazole, thiazine, indamine, indophenol, azine, oxazine, anthraquinone, phthalocyanine, or any combination thereof.

[0225] Biological control agents are broadly defined as microorganisms that can be used instead of synthetic pesticides or fertilizers. When the composition includes a biological control agent, the biological control agent can comprise 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, a lipopeptide-producing Bacillus subtilis strain, a lipopeptide-producing Bacillus amyloliquefaciens strain, a Bacillus firmus strain or a Bacillus pumilus strain.

[0226] When the composition includes a fertilizer, the fertilizer can include ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcitic limestone, calcium oxide, Attorney Docket No. 103362-028WO1 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 nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, urea ammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, K2SO4-Mg2SO4, kainite, sylvinite, kieserite, Epsom salts, elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fish manure, blood meal, rock phosphate, super phosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, humic acid, or any combination thereof.

[0227] The fertilizer can comprise a liquid fertilizer or a dry fertilizer. The composition can include a micronutrient fertilizer material, the micronutrient fertilizer material comprising boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, a sodium tetraborate decahydrate, an iron sulfate, an iron oxide, iron ammonium sulfate, an iron frit, an iron chelate, a manganese sulfate, a manganese oxide, a manganese chelate, a manganese chloride, a manganese frit, a sodium molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinc carbonate, a zinc frit, zinc phosphate, a zinc chelate, or any combination thereof.

[0228] When the composition includes a biostimulant, the biostimulant can comprise a seaweed extract, an elicitor, a polysaccharide, a monosaccharide, a protein extract, a soybean extract, a humic acid, a plant hormone, a plant growth regulator, or any combination thereof.

[0229] Examples of thickeners (i.e., compounds that impart a modified flowability to formulations (i.e., high viscosity under static conditions and low viscosity during agitation) are polysaccharides and organic and inorganic clays such as Xanthan gum (Kelzan®, CP Kelco, U.S.A.), Rhodopol® 23 (Rhodia, France), Veegum® (R.T. Vanderbilt, U.S.A.) or Attaclay® (Engelhard Corp., NJ, USA).

[0230] Examples of suitable anti-freezing agents are ethylene glycol, propylene glycol, urea, and glycerin.

[0231] Examples of anti-foaming 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.

[0232] The fungicide can comprise aldimorph, ampropylfos, ampropylfos potassium, andoprim, anilazine, azaconazole, azoxystrobin, benalaxyl, benodanil, benomyl, benzamacril, benzamacryl-isobutyl, benzovindflupyr, bialaphos, binapacryl, biphenyl, Attorney Docket No. 103362-028WO1 bitertanol, blasticidin-S, boscalid, bromuconazole, bupirimate, buthiobate, calcium polysulphide, capsimycin, captafol, captan, carbendazim, carvon, quinomethionate, chi obenthi azone, chlorfenazole, chloroneb, chloropicrin, chlorothalonil, chlozolinate, clozylacon, cufraneb, cymoxanil, cy proconazole, cyprodinil, cyprofuram, debacarb, dichlorophen, diclobutrazole, diclofluanid, diclomezine, dicloran, diethofencarb, dimethirimol, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinocap, diphenylamine, dipyrithione, ditalimfos, dithianon, dodemorph, dodine, drazoxolon, edifenphos, epoxiconazole, etaconazole, ethirimol, etridiazole, famoxadon, fenapanil, fenarimol, fenbuconazole, fenfuram, fenitropan, fenpiclonil, fenpropidin, fenpropimorph, fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, fludioxonil, flumetover, fluoromide, fluoxastrobin fluquinconazole, flurprimidol, flusilazole, flusulfamide, flutolanil, flutriafol, folpet, fosetyl-aluminium, fosetyl-sodium, fthalide, fuberidazole, furalaxyl, furametpyr, furcarbonil, furconazole, furconazole-cis, furmecyclox, guazatine, hexachlorobenzene, hexaconazole, hymexazole, imazalil, imibenconazole, iminoctadine, iminoctadine albesilate, iminoctadine triacetate, iodocarb, iprobenfos (IBP), iprodione, irumamycin, isoprothiolane, isovaledione, kasugamycin, kresoxim-methyl, copper preparations, such as: copper hydroxide, copper naphthenate, copper oxychloride, copper sulphate, copper oxide, oxine-copper and Bordeaux mixture, mancopper, mancozeb, maneb, meferimzone, mepanipyrim, mepronil, metconazole, metalzxyl, methasulfocarb, methfuroxam, metiram, metomeclam, metsulfovax, mildiomycin, myclobutanil, myclozolin, nickel dimethyldithiocarbamate, nitrothal -isopropyl, nuarimol, ofurace, oxadixyl, oxamocarb, oxolinic acid, oxycarboxim, oxyfenthiin, paclobutrazole, pefurazoate, penconazole, pencycuron, phosdiphen, picoxystrobin, pimaricin, piperalin, polyoxin, polyoxorim, probenazole, prochloraz, procymidone, propamocarb, propanosine-sodium, propiconazole, propineb, prothiocinazole, pyrazophos, pyrifenox, pyrimethanil, pyroquilon, pyroxyfur, quinconazole, quintozene (PCNB), a strobilurin, sulphur and sulphur preparations, tebuconazole, tecloftalam, tecnazene, tetcyclasis, tetraconazole, thiabendazole, thicyofen, thifluzamide, thiophanate-methyl, tioxymid, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazbutil, a triazole, triazoxide, trichlamide, tricyclazole, triclopyr, tridemorph, trifloxystrobin, triflumizole, triforine, uniconazole, validamycin A, vinclozolin, viniconazole, zarilamide, zineb, ziram and also Dagger G, OK-8705, OK-8801, a-(l,l- dimethylethyl)-(3-(2 -phenoxy ethyl)-lH-l, 2, 4-triazole-l -ethanol, a-(2,4-dichlorophenyl)-[3- fluoro-3-propyl-lH-l,2,4-triazole-l -ethanol, a-(2,4-dichlorophenyl)-[3-methoxy-a-methyl- Attorney Docket No. 103362-028WO1

[0233] 1H-1, 2, 4-triazole-l -ethanol, a-(5-methyl-l,3-dioxan-5-yl)-[3-[[4-(trifluoromethyl)-phenyl]- methylene]-lH-l,2,4-triazole-l -ethanol, (5RS,6RS)-6-hydroxy-2,2,7,7-tetramethyl-5-(lH- 1 ,2,4-triazol- 1 -y l)-3 -octanone, (E)-a-(methoxyimino)-N-methyl -2 -phenoxy- phenyl acetamide, 1 -isopropyl {2-methyl-l-[[[ l-(4-methylphenyl)-ethyl]-amino]-carbonyl]- propyl } carbamate, 1 -(2,4-dichlorophenyl)-2-( 1H- 1 ,2,4-triazol- 1 -yl)-ethanone-O-(phenyl methyl)-oxime, l-(2-methyl-l -naphthal enyl)-lH-pyrrole-2, 5-dione, l-(3,5-dichlorophenyl)- 3-(2-propenyl)-2,5-pyrrolidindione, l-[(diiodomethyl)-sulphonyl]-4-methyl -benzene, 1 -[[2- (2,4-dichlorophenyl)-l, 3-dioxolan-2-yl]-methyl]-lH-imidazole, l-[[2-(4-chlorophenyl)-3- phenyloxiranyl]-methyl]-lH-l,2,4-triazole, l-[l-[2-[(2,4-dichlorophenyl)-methoxy]- phenyl]-ethenyl]-lH-imidazole, l-methyl-5-nonyl-2-(phenylmethyl)-3-pyrrolidinole, 2', 6'- dibromo-2-methyl-4'-trifluoromethoxy-4'-trifluoro-methyl-l, 3-thiazole-carboxanilide, 2,2- dichloro-N-[l-(4-chlorophenyl)-ethyl]-l-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-[(l-methylethyl)-sulphonyl]-5- (trichloromethyl)-l,3,4-thiadiazole, 2-[[6-deoxy-4-O-(4-O-methyl-(3-D-glycopyranosyl)-a- D-glucopyranos yl]-amino]-4-methoxy-lH-pyrrolo[2,3-d]pyrimidine-5-carbonitrile, 2- aminobutane, 2-bromo-2-(bromomethyl)-pentanedinitrile, 2-chl oro-N-(2, 3 -dihydro- 1, 1,3- trimethyl-lH-inden-4-yl)-3-pyridinecarboxamide, 2-chl oro-N-(2,6-dimethylphenyl)-N- (isothiocyanatomethyl)-acetamide, 2-phenylphenol (OPP), 3,4-dichloro-l-[4- (difluoromethoxy)-phenyl]-pyrrole-2,5-dione, 3,5-dichloro-N-[cyano[(l-methyl-2- propynyl)-oxy]-methyl]-benzamide, 3 -(1 , 1 -dimethylpropyl- 1 -oxo- lH-indene-2-carbonitrile, 3-[2-(4-chlorophenyl)-5-ethoxy-3-isoxazolidinyl]-pyridine, 4-chloro-2-cyano-N,N- dimethyl-5-(4-methylphenyl)-lH-imidazole-l -sulphonamide, 4-methyl-tetrazolo[l,5- a]quinazolin-5(4H)-one, 8-(l,l-dimethylethyl)-N-ethyl-N-propyl-l,4-dioxaspiro[4, 5]decane-2-methanamine, 8-hydroxyquinoline sulphate, 9H-xanthene-2-[(phenylamino)- carbonyl]-9-carboxylic hydrazide, bis-(l-methylethyl)-3-methyl-4-[(3-methylbenzoyl)- oxy]-2,5-thiophenedicarboxylate, cis-l-(4-chlorophenyl)-2-(lH-l,2,4-triazol-l-yl)- cycloheptanol, cis-4-[3-[4-(l,l-dimethylpropyl)-phenyl-2-methylpropyl]-2,6-dimethyl- morpholine hydrochloride, ethyl [(4-chlorophenyl)-azo] -cyanoacetate, potassium bicarbonate, methanetetrathiol -sodium salt, methyl l-(2,3-dihydro-2,2-dimethyl-inden-l- yl)-lH-imidazole-5-carboxylate, methyl N-(2,6-dimethylphenyl)-N-(5-isoxazolylcarbonyl)- DL-alaninate, methyl N-(chloroacetyl)-N-(2,6-dimethylphenyl)-DL-alaninate, N-(2,3- Attorney Docket No. 103362-028WO1 di chi oro-4-hydroxyphenyl)-l -methyl -cyclohexanecarboxamide, N-(2,6-dimethyl phenyl)-2- methoxy-N-(tetra hydro-2-oxo-3-furanyl)-acetamide, N-(2,6-dimethyl phenyl)-2-methoxy- N-(tetrahydro-2-oxo-3-thienyl)-acetamide, N-(2-chloro-4-nitrophenyl)-4-methyl-3-nitro- benzenesulphonamide, N-(4-cyclohexylphenyl)-l,4,5,6-tetrahydro-2-pyrimidinamine, N-(4- hexylphenyl)-l,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-l-[(chloroacetyl)-amino]-ethyl]-benzamide, N-[3-chloro-4,5-bis(2-propinyloxy)-phenyl]-N'-methoxy-methanimidamide, N-formyl-N- hydroxy-DL-alanine-sodium salt, 0,0-diethyl [2-(dipropylamino)-2-oxoethyl]- ethylphosphoramidothioate, O-methyl S-phenyl phenylpropylphosphoramidothioate, S- methyl l,2,3-benzothiadiazole-7-carbothioate, and spiro[2H]-l-benzopyrane-2,l'(3'H)- isobenzofuran]-3'-one, N-trichloromethyl)thio-4-cyclohexane-l,2-dicarboximide, tetramethylthioperoxy dicarbonic diamide, methyl N-(2,6-dimethylphenyl)-N- (methoxyacetyl)-DL-alaninate, 4-(2,2-difluoro-l,3-benzodioxol-4-yl)-l-H-pyrrol-3- carbonitril, or any combination thereof.

[0234] The strobilurin fungicide can comprise a Strobilurin A, a Strobilurin B, a Strobilurin C, a Strobilurin D, a Strobilurin E, a Strobilurin F, a Strobilurin G, a Strobilurin H, an Azoxystrobin, a Trifloxystrobin, a Kresoxim methyl, a Fluoxastrobin, Picoxystrobin, or any combination thereof.

[0235] The strobilurin fungicide can comprise a non-naturally occurring strobilurin fungicide such as an Azoxystrobin, a Trifloxystrobin, a Kresoxim methyl, a Fluoxastrobin, or any combination thereof. For example, the strobilurin fungicide can comprise a Trifloxystrobin, Fluoxastrobin or Picoxystrobin. Strobilurin fungicides are used to control a range of fungal diseases, including water molds, downy mildews, powdery mildews, leaf spotting and blighting fungi, fruit rotters, and rusts. They are useful for treating a variety of crops, including cereals, field crops, fruits, tree nuts, vegetables, turfgrasses, and ornamentals.

[0236] The triazole fungicide can comprise prothioconazole, imidazole, imidazil, prochloraz, propi conazole, triflumizole, diniconazole, flusilazole, penconazole, hexaconazole, cyproconazole, myclobutanil, tebuconazole, difenoconazole, tetraconazole, fenbuconazole, epoxiconazole, metconazole, fluquinconazole, tri ti conazole, or any combination thereof. Attorney Docket No. 103362-028WO1

[0237] In addition, the fungicide can comprise azoxystrobin, carboxin, difenoconazole, fludioxonil, fluxapyroxad, ipconazole, mefenoxam, pyraclostrobin, silthiofam, sedaxane, thiram, triticonazole or any combination thereof.

[0238] The herbicide can comprise 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulfuron methyl bensulide, bentazon, bispyribac sodium, bromacil, bromoxynil, butylate, carfentrazone, chlorimuron, 2-chlorophenoxy acetic acid, chlorsulfuron, chlorimuron ethyl, clethodim, clomazone, clopyralid, cloransulam, CMPP-P-DMA, cycloate, DCPA, desmedipham, dicamba, dichlobenil, diclofop, 2,4-dichlorophenol, dichlorophenoxyacetic acid, dichlorprop, dichlorprop-P, diclosulam, diflufenzopyr, dimethenamid, dimethyl amine salt of 2,4- dichlorophenoxyacetic acid, diquat, diuron, DSMA, endothall, EPTC, ethalfluralin, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fluorxypyr 1-methyleptylester, fomesafen, fomesafen sodium salt, foramsulfuron, glufosinate, glufosinate-ammonium, glyphosate, halosulfuron, halosulfuron-methyl, hexazinone, 2-hydroxyphenoxy acetic acid, 4-hydroxyphenoxy acetic acid, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaben, isoxaflutole, lactofen, linuron, mazapyr, MCPA, MCPB, mecoprop, mecoprop-P, mesotrione, metolachlor-s, metribuzin, metsulfuron, metsulfuron-methyl, molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, phenmedipham, picloram, prim isulfuron, prodiamine, prometryn, pronamide, propanil, prosulfuron, pyrazon, pyrithiobac, pyroxasulfone,quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thifensulfuron-methyl, thiobencarb, tralkoxydim, triallate, triasulfuron, tribenuron, tribernuron-methyl, triclopyr, trifluralin, triflusulfuron, or any combination thereof.

[0239] When the composition includes a nematicide, the nematicide can comprise Bacillus firmus, fluopyram, antibiotic nematicides such as abamectin; carbamate nematicides such as acetoprole, Bacillus chilonosporus. chloropicrin, benclothiaz, benomyl, Burholderia cepacia, carbofuran, carbosulfan, and cleothocard; dazomet, DBCP, DCIP, alanycarb, aldicarb, aldoxycarb, oxamyl, diamidafos, fenamiphos, fosthietan, phosphamidon, cadusafos, chlorpyrifos, diclofenthion, dimethoate, ethoprophos, fensulfothion, fostiazate, harpins, heterophos, imicyafos, isamidofos, isazofos, methomyl, mecarphon, Myrothecium verrucaria, Paecilomyces lilacinus, Pasteuria nishizawae (including spores thereof), Attorney Docket No. 103362-028WO1 phorate, phosphocarb, terbufos, thionazin, triazophos, tioxazafen, dazomet, 1,2- dicloropropane, 1,3 -di chloropropene, furfural, iodomethane, metam, methyl bromide, methyl isothiocyanate, xylenol, or any combination thereof. For example, the nematicide can comprise Bacillus firmus strain i-2580, Pasteuria nishizawae (including spores thereof), or fluopyram.

[0240] When the composition includes a bacteriocide, the bacteriocide can comprise streptomycin, penicillins, tetracyclines, oxytetracycline, kasugamycin, ampicillin, oxolinic acid, chlorotetracycline, copper oxide, or any combination thereof. For example, the bacteriocide can comprise oxytetracycline.

[0241] When the composition includes an insecticide, the insecticide can comprise clothianidin, imidacloprid, an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or any combination thereof. For example, the insecticide can comprise clothianidin or imidacloprid.

[0242] When the composition includes an insecticide, the insecticide can include an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or any combination thereof.

[0243] Also disclosed are kits that comprise a compound disclosed herein in one or more containers. The disclosed kits can optionally include agriculturally acceptable carriers and / or diluents. In one embodiment, a kit includes one or more other components, auxiliaries, or adjuvants as described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a compound and / or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, a compound and / or agent disclosed herein is provided in the kit as a liquid or solution.

[0244] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. Attorney Docket No. 103362-028WO1

[0245] EXAMPLES

[0246] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0247] 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 indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0248] Design, Expression, and Purification of MTDs. Human Fibronectin Type III (FN3) domain was chosen as the scaffold for Membrane Translocation Domains (MTDs) (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), highly stable protein and has been widely used to develop monobodies that bind to target proteins with high affinity and specificity (Chandler, P.G., et al. (2020) Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain. Cells 9(3):610). Previous studies have demonstrated that several loop regions of FN3 are tolerant to mutations (Steven, A., et al. (2012) Design of novel FN3 domains with high stability by a consensus sequence approach, Protein Engineering, Design and Selection, 25(3): 107-117). Additionally, FN3 is free of any cysteine or disulfide bond and is thus stable in the intracellular environment. FN3 readily folds into its native form without any physical or chemical assistance and can be produced n Escherichia coli in high yields. Finally, FN3 is derived from an abundant human extracellular protein and is less likely to elicit any immune response.

[0249] The BC, DE, and FG loops of FN3 have previously shown to be highly tolerant to sequence mutations. The GDSPAS (SEQ ID NO: 106) sequence of the FG loop was replaced with RRRWWW (SEQ ID NO: 104) to give MTD1 (Table 4). Together with an arginine residue already in the FG loop, this generates a putative CPP motif (R4W3) without altering the loop size. Similarly, the tetrapeptide AVTV of the BC loop was replaced with WWWRRR (SEQ ID NO: 105) to take advantage of the existing arginine in the loop to Attorney Docket No. 103362-028WO1 form a putative CPP, W3R4 (Table 4). The size of the BC loop in the resulting mutant, MTD2, is increased by 2 residues. To explore the possibility of grafting a CPP motif to the other end of FN3, the CPP motif R4W3 was substituted for the tripeptide NSP in the CD loop to give MTD3. To test the feasibility of grafting a CPP sequence into two different loops, a relatively hydrophobic tripeptide in the BC loop (VTV) was replaced with WYW and a hydrophilic motif in the FG loop (GDSPAS; SEQ ID NO: 106) with RRR to produce MTD4. Finally, MTD5 was generated by switching the WYW and RRR motifs of MTD4. Two mutants, MTD4a and MTD4b, which contain 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 previously been reported as the “endosomal escape motif’ of cell-permeable 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 by an online program, Phyre2, to predict their folded structures based on homology of sequences. All mutants maintained a similar overall folding to wild type FN3, with the CPP motifs displayed on their surfaces and constrained into the “cyclic” topology.

[0250] 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 4). The total cellular entry efficiency of MTD6-10 was assessed by labeling the MTDs at a unique C-terminal cysteine with tetramethylrhodamine-5-maleimide (TMR). HeLa cells were treated with the TMR-labeled proteins (5 pM) for 2 h and analyzed by live cell confocal microscopy. MTD7TMRand MTD9TMRshowed similar uptake as MTD4TMR, whereas MTD6TMR, MTD8TMRand MTD10TMRshowed much less cellular entry. Additionally, the isolated yields for MTD6-10 varied from 0.6 to 6.2 mg / L of E. coli cell culture (Table 3). Note that MTD4 and MTD6 differ only slightly in the BC loop sequence (“WYW” vs “YWW”) and yet have dramatic differences in the isolated yields (9.4 mg / L vs 0.9 mg / L) as well as the cell entry efficiency. Similarly, swapping the CPP motifs between the BC and FG loops of MTD4 resulted in a poorly expressed and much less active variant (MTD5 in Table 4). These results demonstrate that the proper folding / stability and high cellular entry efficiency of the MTDs require not only the presence of amphipathic CPP motifs but also their proper presentation on the protein surface. Attorney Docket No. 103362-028WO1

[0251] Table 3. List of strains and plasmids used in study.

[0252] The DNA sequence coding for WT FN3 was chemically synthesized and ligated into prokaryotic expression vector pET-15b. To facilitate protein purification and genetic 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) followed by a recognition site for restriction endonuclease SacI and a cysteine was added to its C-terminus (Table 5). All loop insertion mutants were generated by 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.

[0253] Virol. Meth. 149:85-9020) and expressed in E. coli. Among the mutant proteins, MTD1 failed to produce significant amounts of soluble protein, whereas WT FN3 and MTD2-5 produced soluble proteins in good yields (Table 4). All proteins were purified to near homogeneity by metal affinity chromatography on a Ni-NTA column.

[0254] Table 4. Loop Sequences and Expression Yields of MTDs Attorney Docket No. 103362-028WO1

[0255] *Underlined residues were deleted and bold-faced residues were inserted during mutagenesis.

[0256] Cloning, Expression, and Purification of MTDs. All loop insertion mutants were generated by one-step polymerase-chain reaction (PCR) method (Qi, D., ztZ.). The peptide sequence (Table 5) for each construct was confirmed by sequencing the entire coding region of the plasmid DNA. Pilot-scale protein expression was carried out to check the levels of expression for Mutant proteins, all mutants were expressed in 5 mL E. coli BL21 (DE3) bacterial culture. The induction was carried out in presence of 0.25 mM IPTG at 37 °C. The level of expression was checked by comparing total cell lysate before and after induction on SDS gel. Attorney Docket No. 103362-028WO1

[0257] Table 5. Amino Acid Sequences of WT FN3, MTDs, and MTD-Cargo Fusions

[0258] Protein SEQ ID Sequence

[0259] NO:

[0260] FN3 118 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSL

[0261] LISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATI SGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGGSGGS GGSELC

[0262] MTD1 119 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSL

[0263] LISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATI SGLKPGVDYTITVYAVTGRRRRWWWSKPISINYRTGGSG GSGGSELC

[0264] MTD2 120 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSL

[0265] LISWDAPWWWRRRRYYRITYGETGGNSPVQEFTVPGSK STATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGG SGGSGGSELC

[0266] MTD3 121 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSL

[0267] LISWDAPAVTVRYYRITYGETGGRRRRWWWVQEFTVPG SKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT GGSGGSGGSELC

[0268] MTD4 122 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSL

[0269] LISWDAPAWYWRYYRITYGETGGNSPVQEFTVPGSKSTA TISGLKPGVDYTITVYAVTGRRRRSKPISINYRTGGSGGSG GSELC

[0270] MTD4a 123 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTS

[0271] LLISWDAPAWYWRYYRITYGETGGNSPVQEFTVPGSKST ATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGGS GGSGGSELC

[0272] MTD4b 124 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTS

[0273] LLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTA TISGLKPGVDYTITVYAVTGRRRRSKPISINYRTGGSGGS GGSELC

[0274] MTD4c 172 MGSSHHHHHHSSGHMVSDVPRDLEVVAATPTSLLISWD

[0275] APAWYWRYYRITYGETGGNSPVQEFTVPGSKSTATISGL KPGVDYTITVYAVTGRRRRSKPISINYTTGGSGGSGGSEL C

[0276] MTD5 125 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTS

[0277] LLISWDAPARRRRYYRITYGETGGNSPVQEFTVPGSKST ATISGLKPGVDYTITVYAVTGWYWRSKPISINYRTGGSG GSGGSELC

[0278] MTD4- 127 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTS

[0279] N21 LLISWDAP AWYWRYYRIT YGETGGNSP VQEFTVPGSKST

[0280] ATISGLKPGVDYTITVYAVTGRRRRSKPISINYRTGGSGGS GGSELNQGISEKQLDQLLCQLISALL Attorney Docket No. 103362-028WO1

[0281] MTD6 167 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTS

[0282] LLISWDAPAYWWRYYRITYGETGGNSPVQEFTVPGSKST ATISGLKPGVDYTITVYAVTGRRRRSKPISINYRTGGSGG SGGSELC

[0283] MTD7 168 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTS

[0284] LLISWDAPAWWARYYRITYGETGGNSPVQEFTVPGSKST ATISGLKPGVDYTITVYAVTGRRRRSKPISINYRTGGSGG SGGSELC

[0285] MTD8 169 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTS

[0286] LLISWDAPAWWWRRYYRITYGETGGNSPVQEFTVPGSK STATISGLKPGVDYTITVYAVTGRRASSKPISINYRTGGSG GSGGSELC

[0287] MTD9 170 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTS

[0288] LLISWDAPAWWARYYRITYGETGGNSPVQEFTVPGSKST ATISGLKPGVDYTITVYAVTGWRRRRSKPISINYRTGGSG GSGGSELC

[0289] MTD10 171 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTS

[0290] LLISWDAPAWWRRYYRITYGETGGNSPVQEFTVPGSKST ATISGLKPGVDYTITVYAVTGRRWWSKPISINYRTGGSG GSGGSELC bMTD4- 184 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEIVAATPTSL

[0291] N21 LISWDAP AWYWRYYRIT YGETGGS SP VQEFTVPGSKST A

[0292] TISGLKPGVDYTITVYAVTGRRRRSKPVSINYRTGGSGGS

[0293] GGSELNQGISEKQLDQLLCQLISALL

[0294] The large-scale expression conditions were the same as used for small scale, E. coli cells were centrifuged and stored at -80 °C. These cells were lysed using lysis buffer (50 mL of wash buffer, 0.2 mg / mL lysozyme, 2 mM P-mercaptoethanol, 2 mM PMSF, 2 tablets of Roche complete protease inhibitor cocktail). After homogeneously resuspending the cell palate in lysis buffer the cells were sonicated (Amp. 70%) twice. The crude cell lysate was centrifuged (12000g for 20 min) and the soluble cell lysate was collected. Protein purification was carried out by using fast protein liquid chromatography (FPLC) and the soluble cell lysate was loaded onto aNi-NTA column (with 15 mM imidazole). The column was exhaustively washed with wash buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 5% glycerol and 50 mM imidazole). Protein was eluted with wash buffer containing a linear gradient of 50-500 mM imidazole (pH 7.4) over 30 min.

[0295] MTD4 Plasmid construction. The DNA sequences coding for the tenth FN3 domain and HrpZ were chemically synthesized and ligated with prokaryotic expression vector pET-15b (MilliporeSigma), which had been linearized by using restriction Attorney Docket No. 103362-028WO1 endonucleases Ndel and A / ml. The cloning procedure also resulted in the addition of a flexible linker sequence, (GGS)s, a restriction site (Sad), and a cysteine residue to the C- terminus of FN3. Site-directed mutagenesis of the FN3 to generate plasmid pET-15b-MTD4 was 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.

[0296] J. Virol. Methods 2008, 149 (1), 85-90). The HrpZ gene was amplified by PCR using plasmid DNA as template and primers containing Sad and Xhol restriction sites at the 5’ and 3’ terminus of the HrpZ sequence, respectively (Table 6). The PCR product was digested with Sad and Xhol restriction enzymes and ligated into plasmid pET-15b-MTD4 linearized with the same two enzymes. All the constructs comprise a six-histidine tag at the

[0297] N-terminus for facile purification. The authenticity of the DNA construct was confirmed by restriction mapping and sequencing of the entire coding sequence.

[0298] Table 6. List of primer sequences used in study. Design, expression, and purification of MTD4-N21. The MTD4-N21 fusion protein was generated by recombinantly fusing the N21 peptide from the N-terminal coiled- coil domain of harpin protein Hpal to the C-terminus of MTD4 (Ji et al. 2020). A flexible Attorney Docket No. 103362-028WO1 linker sequence, (GGS)s, was incorporated to minimize any potential interaction between MTD4 and N21. An N-terminal six-histidine tag was added to facilitate protein purification. The fusion protein was expressed in E. coli as inclusion bodies, which were isolated by centrifugation, exhaustively washed, and solubilized in 8 M urea solution. MTD4-N21 was purified to near homogeneity by passing through a Ni-NTA column under denaturing conditions (Figure 1) and refolded by a rapid dilution method.

[0299] MTD4-N21 induces cell death of tomato plants at low concentrations. Four- week-old tomato plants were sprayed with different concentrations of MTD4-N21 (0.6, 0.7, 0.8, 1.0, and 1.5 pM) and chemically synthesized N21 peptide (10, 20, 25, 30, and 50 pM). After 4 days, cell death on the plants was scored. MTD4-N21 caused strong cell death on all treated plants including those sprayed at the lowest concentration (0.6 pM) (Figure 2A). In contrast, no cell death was observed on any of the plants treated with N21 including those sprayed with 50 pM N21 (Figure 2B). Thus, fusion with MTD4 enhances the cell death activity of the N21 peptide by >80-fold, likely as the result of increased cell entry efficiency.

[0300] MTD4-N21 elicits strong plant defense at low concentrations. Total RNAs from MTD4-N21 -treated and control plants (0.03% Tween water) were isolated at 0 and 48 h after the protein spray. qRT-PCR analysis was carried out with DNA primers targeting two defense-related genes, the NAC-containing transcription factor gene (NAC) and the osmotin-like protein gene (OLP) (Pombo, M. A., et al., 2017, Scientific Reports 7: 44905 ("Pombo et al. 2017"); Ma, Q., et al., 2022, Hortic Res 9 ("Ma, et al. 2022")). At 0.6 pM concentration, MTD4-N21 induced similar levels of expression of both genes to those of 25 pM N21 peptide (Figure 3A-3D). These results support cell death data and demonstrate that fusion with MTD4 greatly increases the biological activity of N21 peptide in tomato plants (by 41 -fold).

[0301] MTD4-N21 protects tomato plants against bacterial speck disease caused by Pseudomonas syringae pv. tomato (Pst) DC3000. To determine whether activation of defense gene expression by MTD4-N21 leads to enhanced resistance to pathogens, 4-week- old tomato plants were inoculated with Pst DC3000 (at an optical density of 0.000035) using the vacuum infiltration method of Pombe and colleagues with some modifications (Pombo, M. A., et al., 2014, Genome Biology 15: 492 (“Pombo et al. 2014”)). Plants treated with MTD4-N21 (0.8 pM) showed some small lesions on both the adaxial and abaxial sides of the infected leaves, compared with the control (0.03% Tween water) Attorney Docket No. 103362-028WO1

[0302] (Figures 4A-4D). After destaining the infected leaves to remove the chlorophylls (Anderson, J. C., et al., 2006, Plant Cell 18: 502-514 (“Anderson et al. 2006”), numerous brown spots were readily visible on the leaves of the control plants. In contrast, both the number and the intensity of brown spots were significantly lower on MTD4-N21 treated leaves (Figures 4E-4F). These results demonstrate that MTD4-N21 provides strong protection to tomato plants against the bacterial pathogen. Ji et al. (2020) previously reported that N21 induces host resistance to bacterial and fungal infections in tobacco, tomato, and peach but requires a concentration of 17 pM. 0.8 pM MTD4-N21 is sufficient to protect tomato plants from the Pst infection. These observations suggest that fusion with MTD4 increases the crop-protecting activity of N21 by >20-fold.

[0303] MTD4-N21 induces strong defense gene expression in tomato after Pst DC3000 infection. To unravel the molecular basis of enhanced resistance in MTD4-N21 -treated tomato plants, total RNAs from inoculated plants were isolated at 0, 12, 24, and 48 h after Pst DC3000 inoculation. qRT-PCR analysis was performed to assess the expression levels of two defense-related genes, NAC and LPSE (encoding a lipid particle serine esterase). The expression levels of both genes were 2-3 times higher in MTD4-N21 -treated plants compared to the control plants (0.03% Tween water) at 48 h after inoculation (Figure 5A- 5B). These results suggest that MTD4-N21 triggers defense gene expression in the tomato plant leading to enhanced resistance to DC3000.

[0304] MTD4-N21 promotes tomato growth. To test the growth-promoting activity of MTD4-N21, 3- and 5-week-old tomato plants in a growth chamber were sprayed with 0.5 and 0.7 pM MTD4-N21 solutions, respectively. 0.03% Tween20 / water (TW Con) was used as the control. Plant growth parameters were evaluated 7 weeks after germination. Relative to the TW Con, MTD4-N21 resulted in statistically significant promotion of tomato growth (20-30%) (Fig. 6).

[0305] MTD4-N21 is stable at room temperature. Refolded MTD4-N21 protein solutions were stored at room temperature for 1, 2, 3, and 5 days. These samples, along with a freshly prepared MTD4-N21 solution, were sprayed on 4-week-old tomato plants to test their ability to induce cell death. Cell death lesions on the leaves of the treated plants were recorded as “small” or “large” categories based on the size of the lesions (Table 7). Attorney Docket No. 103362-028WO1

[0306] Table 7. MTD4-N2 solutions (#31, 0.8 pM) freshly prepared, 1, 3 and 5 days kept at RT were used for the protein stability assay. Number of small and big cell death lesions in each treated plant were recorded 5 days after protein spray.

[0307] After 1-5 days of storage at room temperature, MTD4-N21 induced similar levels of cell death compared to the freshly prepared solution (Figs. 7A-7D).

[0308] The stability of MTD4-N21 inclusion bodies upon storage was also assessed. The inclusion bodies were flash frozen and stored at -80 °C before being lyophilized for 48 h. The lyophilized pellet was reduced to a powder and stored at room temperature for varying periods of time (2 weeks, 1 month, 3 months, 6 months, and 1 year). Equal aliquots of the lyophilized inclusion bodies (100 mg each; after storage) were dissolved in buffer A containing 8 M urea, refolded, and tested for biological activity by the cell death assay as previously described. Storage of the inclusion bodies at room temperature for a month did not reduce the activity of the refolded MTD4-N21. The activity of MTD4-N21 after storage for >3 months remained to be determined.

[0309] These findings suggest that MTD4-N21 inclusion bodies remain stable and effective over time, providing the potential for prolonged agricultural use without the need for refrigeration.

[0310] Experimental

[0311] Molecular Cloning. The DNA sequence coding for N21 was chemically synthesized with SacI and Xhol restriction sites installed at the 5’ and 3’ end, respectively. The DNA fragment was ligated into plasmid pET-15b-MTD4 that had been digested with SacI and Xhol restriction enzymes. The DNA sequence of the entire coding region was confirmed by restriction mapping and DNA sequencing to code for the protein sequence SEQ ID NO: 127. Attorney Docket No. 103362-028WO1

[0312] SEQ ID NO: 127:

[0313] MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAWYW RYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRSKPISI NYRTGGSGGSGGSELNQGISEKQLDQLLCQLISALL.

[0314] Expression, Purification, and Refolding of MTD4-N21. Escherichia coli pLysS Rosetta (DE3) cells transformed with plasmid pET15b-MTD4-N21 were grown in 1.5 L of Luria Broth (LB) supplemented with 75 pg / mL ampicillin and 35 pg / mL chloramphenicol at 37 °C until the ODeoo reached 0.6. Protein expression was induced by the addition of 0.25 mM isopropyl B-D-l -thiogalactopyranoside (IPTG) for 6 h at 37 °C. The cells were collected by centrifugation at 5,000 rpm (GS-3 rotor) for 20 min and lysed in 50 mL of buffer A (20 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM beta-mercaptoethanol) supplemented with 2 tablets of Roche complete protease inhibitor cocktail and 0.2 mg / mL lysozyme. The lysate was briefly sonicated and centrifuged at 15,000 rpm (SS-34 rotor) for 25 min to separate the soluble and insoluble fractions.

[0315] The soluble fraction was collected, and ammonium sulfate was added to reach 40% saturation. The mixture was centrifuged at 7,000 rpm (SS-34 rotor) for 20 min and the pellet was discarded. The supernatant containing soluble MTD4-N21 was buffer exchanged into 20 mL of buffer A containing 50% glycerol, and quickly frozen and stored at -80 °C.

[0316] The insoluble fraction (inclusion bodies) was washed with 50 mL of buffer A containing 2% Triton X-100 with two rounds of resuspension and centrifugation. The pellet was solubilized in 25 mL of buffer A containing 8 M urea with constant stirring for 3 h at room temperature. The protein solution was incubated with 1 mL of Ni-NTA resin for 1 h at room temperature with constant stirring. The protein-resin mixture was transferred into a column and the unbound protein solution was drained. The bound protein was eluted by incubating the resin with buffer A containing 8 M urea and varying concentrations of imidazole (50 mM, 250 mM, and 500 mM) for 30 min (with stirring) followed by draining. For the last elution step, buffer A containing 8 M urea and 500 mM imidazole was stirred with the Ni-NTA resin overnight before eluting. The eluted MTD4-N21 fractions were collected and combined, and the concentration was estimated by the Bradford assay. Refolding of MTD4-N21 was carried out by rapidly diluting small aliquots of the 8 M urea solution into 1 L of refolding buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM beta- mercaptoethanol, and 0.6 M arginine) and constantly stirring the solution at room temperature. The addition of the protein solution was carefully controlled so that the protein Attorney Docket No. 103362-028WO1 concentration in the refolding solution increased by 10 pg / mL in every 45 min and to a final concentration of 40 pg / mL. The refolded MTD4-N21 was concentrated by ammonium sulfate precipitation. The 40-80% saturation fraction was collected and resuspended in buffer A containing 50% glycerol. The purity and concentration of MTD4-N21 were determined by SDS-PAGE analysis and by comparison with known amounts of pure MTD4-N21 loaded on the same gel.

[0317] Plant material, Growth conditions and Cell Death Assay. The tomato seeds were initially germinated in vitro using half-liquid Murashige and Skoog media. After one week, the germinated seedlings were transplanted into C20 soil. The plants were subsequently cultivated in a controlled growth chamber under specific conditions: a temperature maintained at 24 ± 2 °C, a photoperiod of 12 h of light and 12 h or dark, humidity set at 65- 80%, and illumination provided by white fluorescent light at an intensity of 180 pmol m“2s"1.

[0318] For the cell death assay, tomato plants were treated with different concentrations of MTD4-N21 (0.6, 0.7, 0.8, 1.0, and 1.5 pM) and synthesized N21 peptide (10, 20, 25, 30, and 50 pM) by foliar spraying. Following treatment, the plants were subjected to high humidity conditions overnight before being transferred to a growth chamber. After a 4-day incubation period, the extent of cell death was assessed based on the presence and size of lesions observed on the leaves. All experiment was conducted with three replicates and the entire process was repeated three times to ensure reliability and consistency of the results.

[0319] Bacterial strain and growth condition. The bacterial strain, Pseudomonas syringae pv. tomato (Pst) strain DC3000 was used. Bacteria was grown on King’s B (KB) medium at 28 °C temperature. The medium was supplemented with rifampicin at a concentration of 25 mg / L to ensure selective growth.

[0320] Pathogen Preparation and Inoculation of Tomato Plants. In accordance with the methodology described by Pombo et al, with some modifications, the process of preparing the pathogen and inoculating tomato plants began by streaking Pst strain DC3000 from glycerol stocks onto KB medium plates supplemented with rifampicin (Pombo et al. 2014). These plates were then placed in an incubator set at 28 °C for a duration of 48 hours to promote optimal bacterial proliferation and growth. For uniform inoculation, a fraction of Pst from glycerol stocks was suspended in liquid KB medium to reach an optical density at 600 nm of 0.1. Then, 0.1 mL of this bacterial suspension was pipetted onto new KB agar plates and left to incubate at 28°C overnight. This procedure was essential for ensuring a Attorney Docket No. 103362-028WO1 consistent bacterial concentration prior to inoculation onto the tomato plants. On the day of infiltration, bacterial colonies were scraped from plates and suspended in 10 mL of sterilized ddH2O and then washed twice by centrifugation at 4000 rpm for 5 min each. To prepare the inoculum, the bacterial cells were resuspended in 10 mM MgCh solution to an optical density at 600 nm of 0.5. This suspension was then used to inoculate 2 liters of 10 mM MgCh solution supplemented with 0.002% silwet, adjusting the final bacterial concentration to an OD600 of 0.000035.

[0321] Germinated tomato plants were transplanted into 4-inch pots and placed in a growth chamber with controlled environmental conditions. After four weeks of growth from seed sowing, the plants were foliar sprayed with 0.08 pM of MTD4-N21 and 0.03% Tween water as a negative control. After 2 days of protein spray, plants were subjected to infiltration with Pst strain DC3000 at a concentration of 3.5 x 103 cfu / mL. This infiltration method involved fully submerging the plants in the bacterial inoculum and applying a 3 min vacuum using a pump set to 0.6 bar. This vacuum step ensured effective and uniform delivery of the bacterial suspension into the plant tissues. Following air-drying, the plants were maintained in high humidity overnight before being transferred to the growth chamber. After 5 days following bacterial infection, disease symptoms were examined using tomato plants treated with 0.03% Tween water as a negative control. For observation, tomato leaves were destained using Carnoy’s fluid, which includes 10% acetic acid, 30% chloroform, and 60% ethanol, following the method outlined by Anderson et al. (2006). The reliability and reproducibility of experimental outcomes were attributed to the consistent management of bacterial strains and plants, alongside the use of a controlled-environment growth chamber, which were identified as key contributing factors.

[0322] RNA Isolation and qRT-PCR expression analysis. Total RNA was extracted from the aerial parts of tomato plant leaves treated with 0.08 pM MTD4-N21 and without protein (0.03% of Tween) using the Direct-zol™ RNA Miniprep kit (Zymo Research, USA) with slight modifications. RNA concentration was measured using a NanoDrop ND- 1000 spectrophotometer (Nanodrop Technologies, USA), and its integrity was assessed via agarose gel electrophoresis. Subsequently, 4 pg of total RNA was used to synthesize cDNA using the HiFiScript gDNA Removal cDNA Synthesis Kit (CoWin Biosciences, China) following the manufacturer's instructions.

[0323] Gene expression profiling of defense pathway-related genes was conducted using specific primers through qRT-PCR analysis. The primer sequences are detailed in Table 8. Attorney Docket No. 103362-028WO1

[0324] All reactions were conducted in triplicate, with the SIARD2 gene serving as the internal control for normalizing gene expression. The selected genes were analyzed using qRT-PCR with Fast SYBR mixture (CoWin Biosciences, China) on a CFX96TM Real-Time System (Bio-Rad, USA). Relative quantification of gene transcripts was performed using the AACT method.

[0325] Protein Stability Assay. To assess the stability of the MTD4-N21, 0.08 pM concentration of protein was prepared and stored at room temperature for 1, 2, 3, and 5 days before being sprayed onto 4-week-old tomato plants. Freshly prepared MTD4-N21 was also applied for comparison. The plants were then transferred to a climate-controlled growth chamber. After 4 days of incubation, the stability of the protein was evaluated by scoring the presence of small and large cell death lesions on the surface of the plant leaves.

[0326] Expression, Purification, and Refolding of Bison Variant MTD4-N21 (bMTD4- N21)

[0327] Non-human FN3 domain as a scaffold for engineering new MTDs. A bison FN3 domain was selected due to its high homology with the human FN3 domain, differing by only three conservative substitutions (Figure 8). This high sequence conservation enabled us to engineer a bison MTD4 variant (bMTD4) by replacing the BC and FG loops of the bison FN3 domain with the cell-penetrating peptide (CPP) motifs WYW and RRRR, respectively, a method previously established for human MTD4 (see PCT Publication No. WO / 2023 / 178327 by Pei, et al).

[0328] The protein and DNA sequences for bMTD4-N21 are provided below.

[0329] Amino Acid Sequence SEQ ID NO. 184

[0330] MGS SHHHHHHS SGLVPRGSHMVSDVPRDLEIVAATPTSLLISWDAP AWYWRYYRI TYGETGGSSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRSKPVSINYRT GGSGGSGGSELNQGISEKQLDQLLCQLISALL

[0331] Nucleic Acid sequence SEQ ID NO. 185

[0332] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGG CAGCCATATGGTCTCAGATGTACCCAGGGACCTAGAAATTGTGGCCGCGACTCC GACCAGCTTGCTGATTTCCTGGGATGCACCGGCGTGGTATTGGCGTTATTACCG CATTACGTACGGCGAAACCGGTGGTAGCAGCCCGGTTCAGGAGTTCACCGTGCC GGGTTCCAAAAGCACCGCTACGATCTCCGGCCTGAAACCGGGTGTAGACTACA CCATCACCGTCTATGCGGTGACCGGCCGTCGTCGACGCAGCAAGCCAGTGAGC ATTAACTATCGTACCGGCGGTTCTGGTGGCTCTGGCGGTTCGGAGCTCAACCAG Attorney Docket No. 103362-028WO1

[0333] GGCATTAGCGAAAAACAGCTGGATCAGCTGCTGTGCCAGCTGATTAGCGCGCT

[0334] GCTGTAA

[0335] Production ofbMTD4-N21. bMTD4-N21 was expressed in A. coli as inclusion bodies, yielding approximately 1.9 g (dry weight) of protein per liter of cell culture. Following a wash with a Triton X-100 buffer and lyophilization, the inclusion bodies were about 90% pure as determined by SDS-PAGE analysis (Figure 9). The protein was then solubilized in a phosphate buffer containing 8 M urea before being refolded through rapid dilution into a refolding buffer.

[0336] Biological Activity ofbMTD4-N21. the biological activity ofbMTD4-N21 was demonstrated by its ability to induce dose-dependent cell death in tomato plants (Figure 10). At higher concentrations (10 and 30 pM), the protein caused a marked increase in the number and size of necrotic lesions compared to the Tween 20 control. In contrast, no visible cell death was observed at the lowest concentration tested (2 pM) (Table 9). The effect of bMTD4-N21 was investigated on the expression of defense-related genes, including NAC, OLP, LPSE, and ACS2 (see Etalo, D.W., et al., 2013. Plant Physiology, 162(3), pp.1599-1617; Ma, Q., et al., 2022. Horticulture Research, 9; Pombo, M.A., et al., 2014. Genome biology, 15(10), p.492; Zhang, N., et al., 2020. Plant physiology, 183(4), pp.1869-1882). A concentration of 10 pM induced the highest level of gene upregulation, indicating a strong activation of defense pathways (Figure 11). However, at 30 pM, despite increased cell death, gene expression levels declined. This suggests that extensive tissue necrosis may suppress further transcriptional responses. Conversely, the 2 pM concentration, which did not cause visible cell death, still upregulated defense genes, implying that low levels of bMTD4-N21 are sufficient to trigger early defense signaling without causing overt cellular damage. Collectively, these findings demonstrate a concentration-dependent balance in tomato leaves: bMTD4-N21 activates defense responses at low concentrations while inducing cell death at higher concentrations.

[0337] Expression and Isolation of Inclusion Bodies. E. coli BL21(DE3) cells transformed with pET15b vector encoding bMTD4-N21 were grown in 1 L of Dynamite media containing 75 pg / mL ampicillin at 37 °C. When the culture reached an ODeoo of 6-8, protein expression was induced by the addition of IPTG (0.25 mM final concentration) and incubation at 37 °C for another 12-14 h. The cell culture was centrifuged at 5,000 rpm for 25 min, and the cell pellet was stored at -80 °C. The cell pellet was resuspended and lysed in 50 mL of KPO4 buffer (10 mM KPO4, pH 7.4, 100 mM KC1) containing 500 pL of halt Attorney Docket No. 103362-028WO1 protease inhibitor (lOOx) and 15 mg of lysozyme followed by brief sonication. The lysate was centrifuged at 14,000 rpm for 25 min, and the inclusion bodies were stored at -80 °C. To remove cell debris and unwanted membrane proteins, the inclusion bodies were washed twice with the KPO4 buffer containing 2% Triton-XIOO. The washed pellet was frozen at - 80 °C and lyophilized for 48 h. The freeze-dried pellet was grounded into a powder before being refolded.

[0338] Refolding of bMTD4-N21. The lyophilized inclusion bodies (20 mg) were solubilized in 1 mL of KPO4 buffer containing 8 M Urea. After 3 h, the solubilized inclusion bodies were slowly added to 500 mL of refolding buffer (10 mM KPO4, pH 7.4, 100 mM KC1, and 200 mM L-arginine) (250 pL protein solution every 45 min). The refolding solution was constantly stirred at room temperature. The final protein concentration in the refolding solution should not exceed 40 pg / mL. Alternatively, 500 mL of the refolding buffer may be directly added to 1 mL of the dissolved inclusion bodies with rigorous stirring, resulting in instant dilution and refolding of bMTD4-N21. The purity and concentration of the refolded protein were assessed by SDS-PAGE, UV-VIS, and a Bradford assay. The molar extinction coefficient of bMTD4-N21 is 26,930 M^cm’1.

[0339] Plant Material, Growth Conditions, and Cell Death Assay. Tomato seeds were first germinated in vitro on half-strength liquid Murashige and Skoog (MS) medium. After one week, the germinated seedlings were transferred to C20 soil. The plants were grown in a controlled-environment growth chamber under standardized conditions: temperature maintained at 24 ± 2 °C, a 12 h light / 12 h dark photoperiod, relative humidity of 65-80%, and illumination with white, fluorescent light at a photosynthetic photon flux density of 180 pmol m“2s"1. For the cell death assay, tomato plants were treated with varying concentrations (2, 10, and 30 pM) of bMTD4-N21, while a control group was treated with 0.03% Tween 20 in water. Treatments were applied via foliar spraying to ensure uniform coverage of the leaves. Following application, the plants were maintained under high humidity conditions overnight to facilitate protein uptake and enhance the induction of cellular responses. The next day, the plants were transferred to a controlled growth chamber, where they were incubated under standard growth conditions for a period of five days. After this incubation, the extent of cell death was carefully evaluated by observing the leaves for the presence, number, and size of necrotic lesions. Lesions were categorized as either small or large to provide a more detailed assessment of the severity of cell death. The experiment was conducted with four biological replicates per treatment, and the entire Attorney Docket No. 103362-028WO1 procedure was independently repeated three times to ensure the reliability and reproducibility of the results.

[0340] RNA Isolation and qRT-PCR Expression Analysis. Total RNA was isolated from the aerial tissues of tomato leaves treated with 2, 10, and 30 pM bMTD4-N21, as well as from control leaves treated with 0.03% Tween 20 in water, using the Direct-zol™ RNA Miniprep Kit (Zymo Research, USA) with minor modifications to the standard protocol to optimize yield and purity. The concentration and purity of the extracted RNA were determined using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, USA), while its integrity and quality were evaluated by agarose gel electrophoresis to confirm the absence of degradation. For downstream gene expression analysis, 1 pg of total RNA from each sample was used to synthesize complementary DNA (cDNA) by using the HiFiScript gDNA Removal cDNA Synthesis Kit (CoWin Biosciences, China) according to the manufacturer’s instructions. This protocol included a genomic DNA removal procedure to ensure that the resulting cDNA was free from genomic contamination, thereby providing reliable templates for subsequent analyses.

[0341] Gene expression analysis of defense-related pathway genes was performed using quantitative real-time PCR (qRT-PCR) with gene-specific primers, whose sequences are provided in Table 8. All qRT-PCR reactions were carried out in triplicate to ensure accuracy and reproducibility of the results. The SIARD2 gene was used as an internal reference to normalize the expression levels of target genes, providing a reliable baseline for comparison across samples. Reactions were prepared using the Fast SYBR Green PCR Master Mix (CoWin Biosciences, China) and run on a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA) under optimized cycling conditions. The relative expression levels of the target transcripts were calculated using the comparative AACT method, allowing precise quantification of gene induction or repression in response to bMTD4-N21 treatment. This approach enabled a detailed assessment of the activation of defense signaling pathways in tomato leaves under different treatment conditions. Attorney Docket No. 103362-028WO1

[0342] Table 8. List of primers used in this study

[0343] Attorney Docket No. 103362-028WO1

[0344] Table 9. Quantitative assessment of cell death based on small and large lesion formation

[0345] SEQUENCES Attorney Docket No. 103362-028WO1 Attorney Docket No. 103362-028WO1 Attorney Docket No. 103362-028WO1 Attorney Docket No. 103362-028WO1 Attorney Docket No. 103362-028WO1 Attorney Docket No. 103362-028WO1

Claims

Attorney Docket No. 103362-028WO1CLAIMSWhat is claimed is:

1. A peptide, comprising: a membrane translocation domain having one or more cell penetrating peptide motifs, and a cargo moiety linked to the membrane translocation domain, wherein the cargo moiety comprises a plant bioactive moiety comprising SEQ ID NO: 126 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126, where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues; or where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least two arginine and / or lysine residues and at least one other cell penetrating peptide motif is from 2 to 8 amino acid residues in length and has at least two hydrophobic residues.

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

3. The peptide of claim 2, wherein the human fibronectin type III has 90% sequence similarity with SEQ ID NO: 118.

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

5. The peptide of any one of claims 1-4, 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 cell penetrating peptide motifs.

6. The peptide of any one of claims 1-5, 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 cell penetrating peptide motifs.Attorney Docket No. 103362-028WO17. The peptide of any one of claims 1-6, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and the BC and either the DE, CD, or FG loops have cell penetrating peptide motifs.

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

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

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

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

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

13. The peptide of any one of claims 1-12, wherein the peptide comprises TGRRRRWWWSKPI (SEQ ID NO: 111); APWWWRRRRYY (SEQ ID NO: 112); GGRRRRWWWVQE (SEQ ID NO: 113); APAWYWRYY (SEQ ID NO: 114); TGRRRRSKPI (SEQ ID NO: 115); APARRRRYY (SEQ ID NO: 116); TGWYWRSKPI (SEQ ID NO: 117); SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, or SEQ ID NO: 165.

14. The peptide of any one of claims 1-13, wherein the peptide comprises SEQ ID NO.s: 119, 120, 121, 122, 123, 124, 125, 127, 167, 168, 169, 170, 171, or 184.Attorney Docket No. 103362-028WO115. The peptide of any one of claims 1-14, wherein the cargo moiety is linked to the membrane translocation domain at a N-terminus or C-terminus of the membrane translocation domain, or at a side chain within the membrane translocation domain.

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

17. The peptide of any one of claims 1-16, wherein the peptide comprises SEQ ID NO: 127 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 127.

18. The peptide of any one of claims 1-16, wherein the peptide comprises SEQ ID NO: 184 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 184.

19. The peptide of any one of claims 1-18, wherein the plant bioactive moiety comprises synthetically derived or naturally occurring flagellins and flagellin-associated polypeptides (including those conserved among the Bacillus genera), thionins, harpin protein or polypeptide or harpin-like polypeptide, elongation factor Tu (EF-Tu), phytosulfokine (PSKa), root hair promoting polypeptide (RHPP), hypersensitive response elicitor proteins or polypeptides, or any combination thereof.

20. A composition for delivering a cargo moiety into a plant cell comprising SEQ ID NO: 122 covalently bound to a cargo moiety, wherein the cargo moiety comprises a plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126.

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

22. A method of delivering a cargo moiety into a plant, comprising contacting the plant with a peptide of any one of claims 1-19 or composition of any one of claims 20-21.

23. A method of delivering a plant bioactive moiety including SEQ ID NO: 126 or variants having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,Attorney Docket No. 103362-028WO198%, 99%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 126 into a plant cell, comprising contacting the plant cell with a peptide of any one of claims 1-19 or composition of any one of claims 20-21.

24. A method of delivering a plant stimulant into a plant, comprising contacting the plant with a peptide of any one of claims 1-19 or composition of any one of claims 20-21.

25. A method of delivering a plant activator into a plant, comprising contacting the plant with a peptide of any one of claims 1-19 or composition of any one of claims 20-21.

26. A method to protect plants against biotic stress; stimulate seeds during germination; to protect plants against abiotic stress; to enhance growth, yield, health, longevity, productivity, and / or vigor of a plant; to provide multiple disease resistance to a plant; or any combination thereof, comprising contacting the plant with a peptide of any one of claims 1-19 or composition of any one of claims 20-21.

27. A method of treating a plant that has a disease caused by a pathogenic agent, comprising contacting the plant with a peptide of any one of claims 1-19 or composition of any one of claims 20-21.

28. The method of claim 27, wherein the pathogenic agent is Pseudomonas syringae pv. tomato.

29. The method of any one of claims 22-28, wherein the plant is a tomato plant.

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