Designed protein compositions for plant uptake and transport

Abstract

This disclosure pertains to the development of proteins that can be used in a spray application to enter plants by moving through the cuticle, make entry to plant cells, and move systemically within the plant. This includes any protein that can be sprayed onto an intact plant and achieve these functions, as well as methods to discover and develop such proteins.

Description

PATENT APPLICATION DOCKET NO. P14953WOOO TITLE: DESIGNED PROTEIN COMPOSITIONS FOR PLANT UPTAKE AND TRANSPORTInventors: Jonathan Lightner, David Long, John JanczyCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 740,159, filed on December 30, 2024, which is hereby incorporated by reference herein in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing P14953WOOO_SequenceListing.xml; Size: 41,686 bytes; Date of Creation: December 18, 2025, is herein incorporated by reference in its entirety.FIELD

[0003] The disclosure is generally related to proteins which can be taken up when applied to intact plants, are mobilized within a plant and methods of use and application thereof BACKGROUND

[0004] Proteins show great promise as biomolecules for crop enhancement and protection, but in order for proteins to have their optimal protective effects, they need to make entry’ into the plant and move systemically within the plant. To make entry into the plant and its cells, proteins must pass through physical and biological barriers that plants have evolved to separate themselves from their environment. Recent advances in protein design, expression, and engineering have created the promise that proteins that are mobilized in the plant, as opposed to other molecules such as chemical pesticides, can be valuable molecules for use in crop production applications. However, challenges associated with penetrating the plant epidermis, such as the cuticle and stomata, represent an obstacle to developing effective protein-based crop inputs.

[0005] Accordingly, there is a need in the art to develop effective methods of introducing proteins into plants such that they enter plant cells and move within the plant.PATENT APPLICATION DOCKET NO. P14953WOOOSUMMARY

[0006] Natural and engineered insecticidal proteins have been created and used in transgenic plants for the control of specific plant pests in major crops such as com, cotton, and soybean. Proteins are also critical modulators of many plant functions.

[0007] However, expressing proteins in transgenic plants suffers from significant challenges. Such plants are difficult to create and cannot be made in all plant systems. Additionally, these approaches are regulated by lengthy processes at the national and international levels, requiring hundreds of millions of dollars to create and market, often taking more than a decade to commercialize.

[0008] Applied chemical pesticides have been more broadly adopted, in part because they can be applied to all plant species. Many have been successful because they enter the plant and move systemically throughout the plant, providing broad benefits beyond the application itself. The instant invention, in some embodiments, teaches proteins, and methods to discover and enhance them, that can enter plant cells when applied as a sprayable solution.

[0009] Small molecule crop protection agents have been developed over time through processes that require that they are successful at entering the plant and its cells. These processes are generally understood in the sense that it is known that certain physical chemical characteristics, like the balance of lipophilicity and hydrophilicity, size, and other attributes are important but it is not at all clear that these same attributes and thinking are applicable to large biomolecules like proteins.

[0010] Plant cell penetrating peptides, plant signaling peptides, and elicitor peptides have been studied to a limited extent, but the emphasis of these efforts has been primarily academic, and primarily in the limited application area of gene transfer (transgenesis and gene editing). As a result, the vast majority of the published work in this sector has been with highly mechanically manipulated systems, such as isolated ‘leaf discs’ (that is, discs cut from a living leaf and floated in solution) or in ‘infiltration’ applications where mechanical force is used to make the initial penetration of the leaf (certainly the cuticle and potentially the cell wall as well). In one of these systems the whole plant is subjected to vacuum, and then pressure, to infiltrate the peptides into leaves (e.g., Fujita et al. 2021. All-Peptide-based polyion complex vesicles: facile preparation and encapsulation of the protein in active form. ACS Polymers Au, 1(1), 30-38). While limited uptake has been shown in this approach, it is not a practical method to make a protein active enter leavesPATENT APPLICATION DOCKET NO. PI4953WOOOin a field or even greenhouse seting. In other approaches, experimental peptides were infiltrated by applying in solution with gentle pressure through a syringe into a supported leaf (also referred to as syringe infiltration, e.g., Xu et al. 2018. Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASEI regulates responses to systemin. necrotrophic fungi, and insect herbivory. The Plant Cell, 30(9), 2214-2229; Terada et al. 2020. Artificial cell -penetrating peptide containing periodic a-aminoisobutyric acid with long-term internalization efficiency in human and plant cells. ACS Biomaterials Science & Engineering, 6(6), 3287-3298). Again, mechanical pressure is responsible for penetrating the leaf cuticle, and again, this approach is obviously not scalable. Further, to the extent these approaches have found cell penetrating peptides, the authors have disclosed that they are species specific and therefore different peptides work on different plants to different degrees (e.g., Numata et al. 2018. Library screening of cell -penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus. Scientific Reports , 8(1), 10966).

[0011] It is known that in some plants (e.g. Tomato and Arabidopsis) small peptides (~<30aa) are generated endogenously within the plant, and are mobilized within the plant to transmit information, for example from root to shoot (in the case of nitrogen status) or from one leaf to another (in the case of wounding) (e.g., Huffaker and Ryan 2007. Endogenous peptide defense signals in Arabidopsis differentially amplify signaling for the innate immune response. Proceedings of the National Academy of Sciences, 104(25), 10732-10736; Ohkubo et al. 2017. Shoot-to-root mobile polypeptides involved in systemic regulation of nitrogen acquisition. Nature plants, 3(4), 1-6; Zhang et al. 2020. Systemin-mediated long-distance systemic defense responses. New Phytologist, 226(6), 1573-1582). While it is known these peptides can move within some plants, few have ever been investigated for their ability7to enter the plant when applied exogenously to an intact leaf, and none have been investigated for the ability to carry’ a functional cargo domain into the plant.

[0012] The present invention relates to compositions, methods, and systems for delivering proteins into plants, for example, through spray application. The invention addresses the need for effective, non-invasive methods to introduce functional proteins into plants for agricultural, horticultural, or biotechnological applications.PATENT APPLICATION DOCKET NO. PI4953WOOO

[0013] The invention aims to combine the utility and engineerability’ of proteins with the properties of uptake and systemic movement that the most successful small molecule crop protection chemicals provide.

[0014] The present invention in some embodiments provides protein sequences, and methods to discover and enhance them, that when applied to intact leaves, enter the plant and move systemically within the plant. The present invention presents combined sequences from cell penetrating peptides and from plant mobile signals (e.g., plant signaling peptides and elicitor peptides) to create new proteins that move systemically in the plant in unexpected ways.

[0015] Further, the instant invention, in some embodiments, further applies novel protein design methodologies to develop and further enhance these movement activities. In some embodiments, the invention provides proteins that can enter the plant when applied as sprayed aqueous solutions to the leaf. These proteins are taken up into plant cells and move systemically through the plant vascular system, accessing multiple parts of the plant.

[0016] By allowing the design of proteins that can enter intact plants when applied as a solution, the instant invention in some embodiments opens all the potential applications of protein actives for crop protection and plant enhancement.

[0017] In some embodiments, the invention includes assays to discover and optimize the activities of such proteins. These assays are designed to identify proteins with desirable properties and enhance their effectiveness in crop protection applications.

[0018] In some embodiments, the invention encompasses methods to improve the function of such proteins. These methods may involve genetic modifications, chemical treatments, or other techniques to enhance the stability, activity’, and systemic movement of the proteins within the plant.

[0019] In some embodiments, the invention provides methods to couple such proteins to other protein actives (also referred to as cargo domains). This coupling allows the additional active proteins to gain access to and move through the plant, thereby extending the benefits of the invention to a broader range of applications.PATENT APPLICATION DOCKET NO. PI4953WOOO

[0020] In one broad aspect, the invention provides a composition comprising proteins capable of penetrating the plant cuticle, entering plant cells, and moving systemically within the plant when applied to an intact plant. These proteins can be naturally occurring, recombinant, or engineered, and may include transport proteins, enzymes, signaling molecules, or proteins with attached cargo domains.

[0021] In another aspect, the invention provides methods of delivering such proteins into plants. These methods involve applying a protein-containing composition, such as a spray formulation, to the surface of an intact plant. The composition may include additional components, such as surfactants, targeting peptides, or stabilizers, to enhance protein efficacy and systemic transport.

[0022] In yet another aspect, the invention includes methods for discovering and developing proteins capable of performing these functions. Candidate proteins are screened for their ability to penetrate plant cuticles, enter plant cells, and move systemically within the plant. Proteins identified through these methods can be further optimized or engineered for enhanced functionality.

[0023] The invention offers significant advantages over existing technologies by providing a non-invasive, versatile, and scalable approach for protein delivery' into plants. Applications of this technology include enhancing plant resistance to abiotic and biotic stresses, delivering nutrients, enabling gene editing, or other biotechnological uses.

[0024] These and other aspects, features, and advantages of the invention will be apparent from the following detailed description and accompanying claims.

[0025] Methods are provided for formulation, use, and application of proteins that are mobilized within a plant. Selected proteins, plants, or parts thereof produced by the aforementioned methods are also provided.

[0026] In some aspects, provided herein is a method of delivering a protein into a plant, plant part, or plant cell, the method comprising contacting the plant, plant part, or plant cell with a composition comprising the protein, the protein comprising a polypeptide selected from a plant signaling peptide and an elicitor polypeptide, wherein the polypeptide is from a source organism that is of a different family, genus, and / or species than the plant, plant part, or plant cell that isPATENT APPLICATION DOCKET NO. P14953WOOOcontacted with the composition, and wherein the protein a) enters plant cells, b) moves within the plant, or c) both (a) and (b). In some embodiments, the protein further comprises a cargo domain.

[0027] In some aspects, provided herein is a method of delivering a protein into a plant, plant part, or plant cell the method comprising contacting the plant with a composition comprising the protein, the protein comprising a HypSys polypeptide, wherein the protein enters the plant and a) enters a plant cell, b) moves within the plant or plant part, or c) both (a) and (b). In some embodiments, the protein further comprises a cargo domain.

[0028] In some aspects, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising a polypeptide selected from the group consisting of a HypSys polypeptide, CEP1 (SEQ ID NO: 1), and ELF18 (SEQ ID NO: 3), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto, wherein the protein enters the plant and a) enters a plant cell, b) moves within the plant or plant part, or c) both (a) and (b). In some embodiments, the protein further comprises a cargo domain. In some embodiments, the elicitor polypeptide is an ELF 18 polypeptide. In some embodiments, the plant signaling polypeptide is a CEP polypeptide, a HypSys polypeptide, a systemin polypeptide, or a PEP polypeptide.

[0029] In some aspects, provided herein is a method of delivering a protein into a plant, the method comprising spraying the leaves of the plant with a composition comprising the protein, the protein comprising a cargo domain and a polypeptide configured to enter the leaves of the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b). In some embodiments, the polypeptide comprises a plant signaling polypeptide and / or an elicitor polypeptide.

[0030] In some aspects, provided herein is a method of delivering a protein into a plant, plant part, or plant cell, the method comprising contacting the plant, plant part, or plant cell with a composition comprising the protein, the protein comprising a cargo domain and a polypeptide configured to enter the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b). wherein the polypeptide comprises a plant signaling peptide and / or an elicitor polypeptide.

[0031] In some aspects, provided herein is a method of delivering a protein into a plant, the method comprising contacting the plant with a composition comprising the protein, the protein comprising a cargo domain and pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%. 70%, 80%, 90%,PATENT APPLICATION DOCKET NO. PI4953WOOOor 95% sequence identity thereto, wherein the cargo domain and pLAA are covalently attached, and wherein the protein enters the plant and a) enters a plant cell, b) moves within the plant, or c) both (a) and (b). In some embodiments, the cargo domain and the pLAA are covalently attached via a triazole ring. In some embodiments, the cargo domain and the pLAA are covalently attached via a peptide bond, optionally comprising a peptide linker between the cargo domain and the pLAA.

[0032] In some embodiments of the foregoing aspects, the contacting comprises contacting the leaves of the plant with the composition. In certain embodiments, the contacting the leaves of the plant with the composition comprises applying one or more droplets of the composition onto the leaves of the plant. In certain embodiments, the contacting the leaves of the plant with the composition comprises spraying the composition onto the leaves of the plant. In certain embodiments, the method comprises pre-treating the leaves of the plant with a pretreatment composition comprising a surfactant prior to contacting or spraying the leaves of the plant with the composition comprising the protein. In some variations, the method comprises spraying the leaves of the plant with the pretreatment composition. In other embodiments of the foregoing aspects, the contacting comprises applying the composition to the roots or to the soil in which the roots are growing.

[0033] In some embodiments of the foregoing aspects, the plant is an intact plant. In some embodiments of the foregoing aspects, the plant is present in an agricultural field.

[0034] In some embodiments of the foregoing aspects, the polypeptide is derived from a source organism that is a different family, genus, and / or species than the species of the plant that is contacted with the composition. In some embodiments, the source organism is a bacterium. In some embodiments, the source organism is a source plant of a different family, genus, and / or species than the plant. In certain embodiments, the source plant is a di cot and the plant is a monocot. In certain embodiments, the source plant is tobacco, tomato, or Arabidopsis thaliana.

[0035] In some embodiments of the foregoing aspects, the plant is a monocot. In certain embodiments, the plant is rye or maize. In other embodiments of the foregoing aspects, the plant is a dicot. In certain embodiments, the plant is Palmer amaranth. In certain embodiments, the plant is Arabidopsis thaliana.PATENT APPLICATION DOCKET NO. P14953WOOO

[0036] In some aspects, provided herein is an isolated protein comprising a cargo domain and a polypeptide configured to enter the leaves of the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b), wherein the polypeptide comprises a plant signaling or elicitor polypeptide. In some embodiments, the plant signaling polypeptide is a CEP polypeptide, a HypSys polypeptide, a systemin polypeptide, or a PEP polypeptide. In some embodiments, the elicitor polypeptide is ELF 18.

[0037] In some embodiments of the foregoing aspects, the polypeptide is between 10 and 40 amino acids in length. In certain embodiments, the polypeptide is between 15 and 35 amino acids in length. In some embodiments, at least 2, 3, 4, 5, 6, 7 or 8 of the residues in the polypeptide are lysine, arginine, and / or histidine residues. In some embodiments, at least 10% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues. In some embodiments, between 10% and 40% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues. In some embodiments, between 20% and 30% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues. In some embodiments, at least 3, 4, 5, or 6 of the residues in the polypeptide are proline and / or hydroxy proline residues. In some embodiments, at least 15% of the amino acids in the polypeptide are proline and / or hydroxyproline residues. In some embodiments, between 15% and 50% of the amino acids in the polypeptide are proline and / or hydroxyproline residues.

[0038] In some embodiments of the foregoing aspects, the systemin polypeptide comprises an amino acid sequence according to Formula (I): AX1X2SX3PPX4KRX5PPKMQTD, wherein XI is V or A; X2 is R, Q, or H; X3 is T or K; X4 is T or S; and X5 or D or P (SEQ ID NO: 15). In certain embodiments, the systemin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 11-14, or an amino acid sequence having at least 60%, 70%. 80%. 90%. or 95% sequence identity thereto.

[0039] In some embodiments of the foregoing aspects, the HypSys polypeptide comprises an amino acid sequence according to Formula (III): X1ZX2X3ZX4X5, wherein each Z is P or O; XI is P, O. S, T, or A; X2 is P, O. S, or T: X3 is P, O. S, T. or A; X4 is K, T. A, or E; and X5 is P. O, S, T, H, K, or Y, wherein O is hydroxyproline. In some embodiments of the foregoing aspects, the HypSys polypeptide comprises an amino acid sequence according to Formula (IV): X1OOX2OX3, wherein O is hydroxyproline; XI is P, O, S, or A; X2 is O, S, T, or A; and X3 is T, K, A, or E. InPATENT APPLICATION DOCKET NO. PI4953WOOOsome embodiments of the foregoing aspects, The method of claim 12, 16, or 17, wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (V): X1PPPX2PX3X4, wherein XI is P, L, or S; X2 is E, S, or A; X3 is K, Q, or E; and X4 is P, K, D, or H (SEQ ID NO: 34). In some embodiments of the foregoing aspects, the HypSys polypeptide comprises an amino acid sequence according to Formula (II): R(Xl)nX2ZX3X4ZX5X6X7X8X9X10Xl 1X12, wherein n is 4, 5, or 6; each Z is P or O; Each XI is A, D, E, G, H, I, K, L, N, P, R, S, T, V, or Y; X2 is P, O, S, T, or A; X3 is P, O, S, or T; X4 is P, O, S, T, or A; X5 is K, T, A, or E; X6 is P, O, S, T, H, K. or Y; X7 is S, D. A, E. or Q; X8 is D, P, O. S, I, A, or no amino acid; X9 is P, O, G, E, I, H, S. or no amino acid; XI 0 is S, Q. T, I, Y. K, N, or no amino acid; XI 1 is K, R, H, N, G, or no amino acid; and XI 2 is Q, P, E, or no amino acid, wherein O is hydroxyproline. In some embodiments, the HypSys polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6 and 16-33, or an amino acid sequence having at least 60%, 70%. 80%, 90%, or 95% sequence identity thereto. In some embodiments, the HypSys polypeptide is a TobHypSys polypeptide. In certain embodiments, the TobHypSys polypeptide is THSI (SEQ ID NO: 5) or THSII (SEQ ID NO: 6), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.

[0040] In some embodiments of the foregoing aspects, the polypeptide is selected from the group consisting of systemin (SEQ ID NO: 4), THSI (SEQ ID NO: 5), THSII (SEQ ID NO: 6), pLAA (SEQ ID NO: 7), pLAAC (SEQ ID NO: 9), CpLAA (SEQ ID NO: 8), CEP1 (SEQ ID NO: 1), PEP1 (SEQ ID NO: 2), and ELF18 (SEQ ID NO: 3), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.

[0041] In some embodiments, provided herein is a recombinant protein comprising a cargo domain and pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA, wherein the cargo domain and the pLAA are covalently attached. In some embodiments, the cargo domain and the pLAA are covalently attached via a triazole ring. In some embodiments, the cargo domain and the pLAA are covalently attached via a peptide bond, optionally comprising a peptide linker between the cargo domain and the pLAA.

[0042] In some aspects, provided herein is a recombinant protein comprising a polypeptide configured to enter the leaves of the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b), the polypeptide comprising: CEP1 (SEQ ID NO: 1), or a polypeptide having atPATENT APPLICATION DOCKET NO. P14953WOOOleast 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1); and pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identify to pLAA, wherein the CEP1 and the pLAA are fused via a peptide bond. In some embodiments, the protein comprises a peptide linker between the CEP1 and the pLAA. In certain embodiments, the peptide linker comprises between 1 and 10 glycine and serine residues. In certain embodiments, the linker is G4S (SEQ ID NO: 10). In some embodiments, the protein comprises, fromN-terminus to C-terminus: CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1); optionally, the peptide linker; and pLAA (SEQ ID NO: 7). or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identify to pLAA. In certain embodiments, the protein comprises CpLAA (SEQ ID NO: 8), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identify to CpLAA (SEQ ID NO: 8). In some embodiments, the protein comprises, from N-terminus to C-terminus: pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%. 70%. 80%, 90%, or 95% sequence identify to pLAA; optionally, the peptide linker; and CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identify to CEP1 (SEQ ID NO: 1). In certain embodiments, the protein comprises is pLAAC (SEQ ID NO: 9), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identify to pLAAC (SEQ ID NO: 9). In some embodiments, the protein further comprises a cargo domain. In certain embodiments, the cargo domain is fused to the N-terminus or the C-terminus of the polypeptide via a peptide bond.

[0043] In some embodiments of the foregoing aspects, the cargo domain and the polypeptide form a complex through non-covalent interactions. In some embodiments of the foregoing aspects, in the cargo domain and the polypeptide are covalently attached. In certain embodiments, the cargo domain and the polypeptide are covalently attached via a peptide bond, optionally comprising a peptide linker between the cargo domain and the polypeptide. In certain embodiments, the cargo domain and the polypeptide are covalently attached via or a triazole ring (click chemistry). In some embodiments, the cargo domain comprises a plant signaling polypeptide, an antigen-binding polypeptide, a site-directed nuclease, a transcription factor, a pesticidal protein, and / or a protein that enhances a plant trait. In certain embodiments, the plant trait is yield, drought tolerance, heat tolerance, frost resistance, nutrient use efficiency, abiotic stress tolerance, or a qualify trait. In some embodiments, the cargo domain is from a different source organism than the plant signaling orPATENT APPLICATION DOCKET NO. PI4953WOOOelicitor polypeptide. In some embodiments, the cargo domain is a recombinant or synthetic polypeptide.

[0044] In some aspects, provided herein is a composition comprising the protein of any one of the foregoing aspects.

[0045] In some embodiments of the foregoing aspects, the composition comprising the protein further comprises a surfactant. In some embodiments of the foregoing aspects, the composition comprising the protein further comprises a pesticide (e.g, an herbicide, a fungicide, and / or an insecticide). In certain embodiments, the composition further comprises glyphosate, glufosinate, 2,4-dichlorophenoxyacetic acid, and / or dicamba. In some embodiments, the composition is a liquid. In some embodiments, rein the composition comprises the protein in an amount of at least about 0.01 mg / mL. In certain embodiments, the composition comprises the protein in an amount of at least about 0.10 mg / mL. In some embodiments, the composition comprises the protein in an amount of between 0.01 mg / mL and 10 mg / mL. In certain embodiments, the composition comprises the protein in an amount of between 0.1 mg / mL and 1 mg / mL. In certain embodiments, the composition comprises the protein in an amount of about 0.1 mg / mL, about 0.25 mg / mL, about 0.5 mg / mL, or about 1 mg / mL.

[0046] In some aspects, provided herein is a plant, plant part, or plant cell comprising the protein of any one of the foregoing aspects. In some embodiments, the plant is an intact plant. In some embodiments, the intact plant is present in an agricultural field. In some embodiments, the polypeptide is derived from a source organism that is a different family, genus, and / or species than the species of the plant, plant part, or plant cell. In certain embodiments, the source organism is a bacterium. In certain embodiments, the source organism is a source plant of a different family, genus, and / or species than the plant, plant part, or plant cell. In certain embodiments, the source plant is a dicot and the plant is a monocot. In certain embodiments, the source plant is tobacco, tomato, or Arabidopsis thaliana. In certain embodiments, the plant is a monocot. In certain embodiments, the plant is ry e or maize. In certain embodiments, the plant is a dicot. In certain embodiments, the plant is Palmer amaranth. In certain embodiments, the plant is Arabidopsis thaliana.PATENT APPLICATION DOCKET NO. P14953WOOODEFINITIONS

[0047] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0048] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0049] The term “isolated” as used herein means having been removed from its natural environment.

[0050] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, part, or parts of the plant. The term “plant” thus includes reference to an immature or mature whole plant, including a plant from which seed or grain or anthers have been removed.

[0051] The term “plant part” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); grain; stover; a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, pods; flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ can be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture can be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. Regenerable cells in a plant cell or tissue culture can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, or stalks. In contrast, some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.

[0052] The term “systemic movement” or “move systemically” includes any intercellular movement within the symplast or apoplast of a plant or plant part. For example, a protein movesPATENT APPLICATION DOCKET NO. PI4953WOOOsystemically within the plant if it moves within or between the vasculature within a leaf, stem, or root. A protein need not be detectable in all parts of the plant or plant part to move systemically within the plant or plant part.

[0053] The term “amino acid substitution” refers to a single amino acid change in a sequence relative to the reference sequence or native sequence. The term “variant” refers to an amino acid sequence having one or more amino acid substitutions relative to the reference or native amino acid sequence.

[0054] The term “pesticide” refers to a compound that kills cells of agricultural pathogens or pests. Pesticides include, for example, herbicides, fungicides, insecticides, nematocides, and bacteriocides. Accordingly, the term “pesticidal proteins” includes, for example, herbicidal proteins, fungicidal proteins, insecticidal proteins, nematocidal proteins, and bacteriocidal proteins.

[0055] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or nonpatent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Several embodiments in which the present disclosure can be practiced are illustrated and described in detail, wherein like reference characters represent like components throughout the several views. The drawings are presented for exemplary purposes and may not be to scale unless otherwise indicated.

[0057] FIG. 1 shows leaf disc results which show the ability of several of the exemplary proteins to enter leaf discs and move throughout the leaf disc.

[0058] FIG. 2A shows the application of several of the exemplary proteins to intact plants and the expansion of movement of the fluorescent proteins from the application point on DI for several peptides into the vasculature of the plant.PATENT APPLICATION DOCKET NO. P14953WOOO

[0059] FIG. 2B shows the application of several of the exemplary proteins to intact plants and the expansion of movement of the fluorescent proteins from the application point on DI for several peptides into the vasculature of the plant.

[0060] FIG. 3 shows evidence for systemic movement with multiple peptides.

[0061] FIG. 4 shows further evidence for systemic movement with additional peptides.

[0062] FIG. 5A depicts a sequence alignment of systemin polypeptides. Sequence alignment was prepared using ClustalW. Asterisk indicates fully conserved residue, colon indicates residues with strong similarity, period indicates residues with weak similarity.

[0063] FIG. 5B depicts a sequence alignment of HypSys polypeptides. ‘"O” indicates a hydroxyproline residue. Sequence alignment was prepared using ClustalW. For sequence alignment with ClustalW, all hydroxyproline residues were converted to P residues for the purposes of aligning the sequences, and were converted back to O residues in the figure. ” / " indicates residues that are either P or O in all aligned sequences.

[0064] FIGS. 6A-6B depict an additional experiment showing penetration of peptides into leaf vasculature and cells in leaf discs. FIG. 6A shows TAMRA fluorescence in six replicate leaf discs incubated with various peptides, a free TAMRA solution, or water. FIG. 6B shows higher magnification images of one representative leaf disc for each of the peptides from the treatments shown in FIG. 6A.

[0065] FIGS. 7A-7C depict an additional experiment showing penetration and movement of peptides in leaves of intact Arabidopsis plants. Peptides were applied to the leaf by dropping 5 pL of solutions containing 2.5 mg / mL of the TAMRA-labeled peptides onto the leaf in several droplets. The droplets were allowed to dry to prevent movement of the peptide on the surface of the leaf. In each figure, the two leftmost columns show BF / fluorescent overlay and fluorescent images of leaves 6d after treatment with the peptides. The columns to the right show selected higher-magnification images of fluorescence in the vasculature where observed at various timepoints after treatment. FIG. 7A shows plants treated with CEP, SYS, THSII, and ELF. FIG.7B shows plants treated with PEP. PLAA. and CPLAA. FIG. 7C shows plants treated with THSI, PLAAC, and water, with exemplary fluorescent trichomes indicated by white arrows.PATENT APPLICATION DOCKET NO. P14953WOOO

[0066] FIGS. 8A-8C depict a third experiment showing penetration and movement of peptides in leaves of intact Arabidopsis plants. Peptides were applied to the leaf by dropping 1 pL of peptide solution at 0.25 mg / mL of the TAMRA-labeled peptides onto the leaf in a single droplet, which was allowed to dry to prevent movement of the peptide on the surface of the leaf. In each figure, the two leftmost columns BF and fluorescent images of leaves 6d after treatment with the peptides. The columns to the right show selected higher-magnification images of fluorescence in the vasculature where observed at various timepoints after treatment. BF images show the application site, which appears as a dark spot after application and drying of the peptide solution. FIG. 8A shows plants treated with CEP, SYS, THSI. and THSII. FIG. 8B shows plants treated with ELF, PEP, CPLAA, and pLAA. FIG. 8C shows plants treated with pLAAC, free TAMRA, and water.

[0067] FIGS.9A-9D depict an experiment showing penetration and movement of ELF and pLAA peptides in leaves of intact Arabidopsis plants when applied at various concentrations. One pL of a Tween-20 solution (1 drop in 1.5 mL) was applied prior to the addition of 1 pL of diluted peptide at the base of the V3 petiole. The peptide solution was briefly allowed to dry in the air before the flat was transferred to a Conviron and imaged after three days. Each figure shows BF (left) and TAMRA fluorescence (right) images of leaves treated at each concentration. BF images show the application site, which appears as a light or dark spot after application and dr ing of the peptide solution, and is indicated with black arrows. FIG. 9A shows untreated leaves. FIG. 9B shows a negative control leaf treated with Tween and water only. FIG. 9C shows leaves treated with various concentrations of ELF. FIG. 9D shows leaves treated with various concentrations of pLAA.

[0068] FIGS. 10A-10I depict an experiment showing penetration and movement of peptides in leaves of intact Palmer amaranth plants. Peptides were applied to the leaf by dropping 1 pL of peptide solution at 1 g / L of the TAMRA-labeled peptides onto the leaf in a single droplet, which was allowed to dry to prevent movement of the peptide on the surface of the leaf. BF images (left) show the application site, which appears as a dark spot (arrows) or streak (brackets) after application and drying of the peptide solution. Fluorescent images (right) were captured Id and 3d after droplet application to show movement of the TAMRA-labeled peptides away from the application site. FIG. 10A shows leaves treated with CEP. FIG. 10B shows leaves treated with ELF. FIG. 10C shows leaves treated with SYS. FIG. 10D shows leaves treated with THSI. FIG.10E shows leaves treated with CPLAA. FIG. 10F shows leaves treated with THSII. FIG. 10GPATENT APPLICATION DOCKET NO. PI4953WOOOshows leaves treated with PEP. FIG. 10H shows leaves treated with pLAAC. FIG. 101 shows leaves treated with pLAA.

[0069] FIGS. 11A-11K depict an additional experiment showing penetration and movement of peptides in leaves of intact Palmer amaranth plants. One pL of a Tween-20 solution (1 drop in 1.5 mL) was applied prior to the addition of 1 pL of diluted peptide onto VI leaves ~7mm from the petiole. The peptide solution was briefly allowed to dry in the air before the flat was transferred to a Conviron and imaged after three days. Each figure shows BF (left) and TAMRA fluorescence (right) images of leaves treated at each concentration. BF images show the application site, which appears as a spot or streak after application and dry ing of the peptide solution, and is indicated with black arrows and brackets, respectively. FIG. 11A shows a first control leaf treated with water.FIG. 11B shows a first leaf treated with water. FIG. 11C shows a control leaf treated with free TAMRA. FIG. 11D shows a leaf treated with CEP. FIG. HE shows a leaf treated with ELF. FIG. HF shows a leaf treated with SYS. FIG. 11G shows a leaf treated with THSII. FIG. 11H shows a leaf treated with PEP.FIG. HI shows a leaf treated with CpLAA. FIG. HJ shows a leaf treated with pLAAC. FIG. HK shows a leaf treated with pLAA.

[0070] FIGS. 12A-12L depict an experiment showing penetration and movement of peptides in leaves of intact cereal rye plants. Peptides were applied to the leaf by dropping 1 pL of peptide solution at 1 g / L of the TAMRA-labeled peptides onto the leaf in a single droplet, which was allowed to dry to prevent movement of the peptide on the surface of the leaf. Since cereal rye leaves are vertical and helical, rye plants were laid on their side and leaves were stabilized with pipette tips for peptide application and imaging. FIG. 12A shows the plants during application and drying (left) and how the leaves were stabilized during peptide application and imaging (right). In FIGS.12B-12L, BF images (left) show the application site, which appears as a dark spot after application and drying of the peptide solution. Fluorescent images (middle / right) of the leaf with the application site were captured 12 h (Day 1) and 3d (Day 3) after droplet application. When fluorescence was detected outside the application site, additional images w ere taken of additional leaf segments in an acropetal direction (tow ard the leaf tip), in w hich case the relative position of the initial and additional leaf segments are show n in a diagram at the right. FIG. 12B shows leaves treated with CEPE FIG. 12C shows leaves treated with SYS. FIG. 12D shows leaves treated with ELF. FIG. 12E show's leaves treated with THSI. FIG. 12F shows leaves treated with THSII. FIG.12G shows leaves treated with PEP. FIG. 12H shows leaves treated with pLAA. FIG. 121 show'sPATENT APPLICATION DOCKET NO. PI4953WOOOleaves treated with CpLAA. FIG. 12J shows leaves treated with pLAAC. FIG. 12K shows leaves treated with free TAMRA. FIG. 12L shows untreated leaves.

[0071] FIGS. 13A-13L depict an additional experiment showing penetration and movement of peptides in leaves of intact r e plants. One pL of a Tween-20 solution (1 drop in 1.5 mL) was applied prior to the addition of 1 pL of diluted peptide at the base of the V3 petiole. The peptide solution was allowed to dry’ before the plants were transferred to a Conviron and imaged after three days. Each figure shows BF (left) and TAMRA fluorescence (right) images of leaves treated at each concentration. When fluorescence was detected outside the application site, additional images were taken of additional leaf segments in an acropetal direction (tow ard the leaf tip). BF images show the application site, which appears as a light or dark spot after application and drying of the peptide solution, and is indicated with black arrows. All images are of the adaxial (upper) surface of the leaf except where indicated otherwise. FIG. 13A shows a control leaf treated with water.FIG. 13B shows a control leaf treated with free TAMRA. FIG. 13C shows a leaf treated with CEP.FIG. 13D shows a leaf treated with SYS. FIG. 13E show s the abaxial (lower) surface of the leaf treated with SYS. FIG. 13F shows a leaf treated with THSI. FIG. 13G shows a leaf treated with THSII. FIG. 13H shows a leaf treated with ELF. FIG. 131 shows a leaf treated with PEP. FIG.13J shows a leaf treated with CpLAA. FIG. 13K shows a leaf treated with pLAAC. FIG. 13L shows a leaf treated with pLAA.

[0072] FIGS. 14A-14F depict an experiment showing penetration and movement of ELF and pLAA peptides in leaves of intact rye plants when applied at various concentrations. One pL of a Tween-20 solution (1 drop in 1.5 mL) was applied prior to the addition of I pL of diluted peptide. The peptide solution was allowed to dry before the plants were transferred to a Conviron and imaged after three days. Each figure shows BF (left) and TAMRA fluorescence (right) images of leaves treated at each concentration. BF images show the application site, which appears as a light or dark spot after application and drying of the peptide solution, and is indicated with black arrows.FIG. 14A shows a control leaf treated with water. FIG. 14B shows leaves treated with various concentrations of ELF. FIG. 14C shows leaves treated with various concentrations of pLAA. FIG.14D shows further images of the leaf treated with ELF at a concentration of 0.5 mg / mL with fluorescence moving distally within the leaf. FIG. 14E depicts linear regression plots of the applied concentration of ELF and pLAA versus the water-normalized mean whole-leaf fluorescence. FIG.PATENT APPLICATION DOCKET NO. P14953WOOO14F depicts plots showing water-normalized fluorescence in leaves treated with ELF and pLAA in the apical and basal direction relative to the application site.

[0073] FIGS. 15A-15D depict an experiment showing penetration and movement of peptides in leaves of maize plants. One pL of a Tween-20 solution (1 drop in 1.5 mL) was applied prior to the addition of 1 pL of diluted peptide (SYS and CEP). The peptide solution was allowed to dry before the plants were transferred to a Conviron and imaged after three days. Each figure shows BF (left) and TAMRA fluorescence (right) images of treated leaves. BF images show the application site, which appears as a light or dark spot after application and drying of the peptide solution, and is indicated with black arrows. FIG. 15A shows a control leaf treated with water. FIG. 15B shows a control leaf treated with free TAMRA. FIG. 15C shows a leaf treated with CEP. FIG. 15D shows a leaf treated w ith SYS.

[0074] FIGS. 16A-16I depict an experiment showing spray application of peptides onto leaves of intact rye plants. FIG. 16A shows a photograph of the experimental setup, in which rye leaves were laid flat and covered with two glass slides, and then sprayed with the peptide solution such that the area between the two glass slides was exposed to the spray. The peptide solution was allowed to dry before the plants were transferred to a Conviron and imaged after three days. FIG.16B shows BF (left) and fluorescence (right) images of leaves of three replicate rye plants treated with water as a negative control. FIG. 16C shows BF (left) and fluorescence (right) images of leaves of three replicate rye plants treated with free TAMRA. FIG. 16D shows BF images of leaves of six replicate rye plants treated w ith systemin. FIG. 16E show s fluorescence images of the same six replicate rye plants treated with systemin. FIG. 16F shows BF images of leaves of five replicate rye plants treated with pLAA. FIG. 16G shows fluorescence images of the same five replicate rye plants treated with pLAA. FIG. 16H show s BF images of leaves of five replicate ry e plants treated with pLAAC. FIG. 161 shows fluorescence images of the same five replicate rye plants treated with pLAAC. White panels indicate no image was taken.

[0075] FIGS. 17A-17V depict an experiment showing spray application of peptides onto leaves of intact Amaranth plants. FIG. 17A shows a photograph of the experimental setup, in which Amaranth leaves w ere laid flat and covered with two glass slides, and then sprayed with the peptide solution such that the area between the two glass slides was exposed to the spray. The peptide solution was allowed to dry' before the plants w ere transferred to a Conviron and imaged after three days. FIG. 17B shows BF (left) and fluorescence (right) images of leaves of two representativePATENT APPLICATION DOCKET NO. PI4953WOOOcontrol Amaranth plants treated with water. FIG. 17C and FIG. 17D show BF (top) and fluorescence (bottom) images of leaves of two representative control Amaranth plants treated with free TAMRA. FIG. 17E, FIG. 17F, FIG. 17G, FIG. 17H, FIG. 171, and FIG. 17J depict BF (top) and fluorescence (bottom) images of leaves of six replicate Amaranth plants treated with systemin.FIG. 17K, FIG. 17L, FIG. 17M, FIG. 17N, FIG. 170, and FIG. 17P depict BF (top) and fluorescence (bottom) images of leaves of six replicate Amaranth plants treated with pLAA. FIG.17Q, FIG. 17R, FIG. 17S, FIG. 17T, FIG. 17U, and FIG. 17V depict BF (top) and fluorescence (bottom) images of leaves of six replicate Amaranth plants treated with pLAAC. White arrows indicate where fluorescence associated with the vasculature is visible outside of the application site.

[0076] An artisan of ordinary skill need not view, within isolated figure(s), the near infinite number of distinct permutations of features described in the following detailed description to facilitate an understanding of the present disclosure.DETAILED DESCRIPTION

[0077] The ability to deliver functional proteins into plants is a critical aspect of modem agricultural, horticultural, and biotechnological practices. Traditional methods of protein delivery’, such as genetic modification and mechanical injection, often face limitations in terms of efficiency, scalability, and invasiveness. There is a growing need for non-invasive, efficient, and scalable methods to introduce proteins into plants to enhance their growth, resistance to diseases, and overall productivity'.

[0078] One promising approach is the use of sprayable protein formulations that can penetrate the plant cuticle, enter plant cells, and move systemically within the plant. This method offers several advantages, including ease of application, reduced labor costs, and minimal damage to the plant. Additionally, sprayable proteins can be used to deliver a wide range of functional proteins, including those that promote growth, enhance resistance to pathogens, and improve stress tolerance.

[0079] Despite the potential benefits, there are challenges associated with developing and optimizing spray able protein formulations. These challenges include ensuring efficient penetration of the plant cuticle, achieving stable and effective protein delivery, and verifying the systemicPATENT APPLICATION DOCKET NO. PI4953WOOOmovement of proteins within the plant. To address these challenges, it is essential to identify and characterize proteins that can effectively penetrate the plant cuticle and move systemically within the plant.

[0080] The present invention provides methods for delivering proteins into plants through spray application and soil application. It also includes methods for discovering and developing such proteins using assays like the intact plant TAMRA assay. These methods offer a non-invasive, efficient, and scalable solution for introducing functional proteins into plants, thereby enhancing their growth, resistance to diseases, and overall productivity.

[0081] The present invention provides a comprehensive approach to delivering functional proteins into plants, offering significant advantages over traditional methods. The sprayable and soil application methods, along with the discovery and development assays, provide a versatile and efficient solution for enhancing plant growth, resistance to diseases, and overall productivity.

[0082] The present invention relates to methods for delivering functional proteins into plants through spray application and soil application. These methods provide a non-invasive, efficient, and scalable approach to enhance plant growth, resistance to diseases, and overall productivity. The invention also includes methods for discovering and developing such proteins using assays like the intact plant TAMRA assay.

[0083] Accordingly, provided herein are methods of delivering proteins to plants, plant parts, or plant cells. In some embodiments, provided herein are methods of delivering proteins to intact plants. In certain embodiments, the plants and plant parts are present in an agricultural setting (e.g. , in a field, greenhouse, or hydroponic system). In some embodiments, the methods comprise contacting the plant with a composition comprising the protein. The contacting may comprise contacting the leaves of the plant (e.g., via a foliar spray), contacting the roots of the plant (e.g., via a soil drench or side dress), or applying the composition to soil prior to the emergence of the plant. In some embodiments, the methods comprise delivering proteins to plant parts or plant cells by contacting plant parts or plant cells with a composition comprising the protein, such as in plant cell culture or plant tissue culture.

[0084] In some embodiments, provided herein is a method of delivering a protein into a plant, plant part, or plant cell, the method comprising contacting the plant, plant part, or plant cell with aPATENT APPLICATION DOCKET NO. PI4953WOOOcomposition comprising the protein, the protein comprising a polypeptide selected from a plant signaling peptide and an elicitor polypeptide, wherein the polypeptide is from a source organism that is of a different family, genus, and / or species than the plant, plant part, or plant cell that is contacted with the composition, wherein the protein a) enters plant cells, b) moves within the plant, or c) both (a) and (b).

[0085] In some embodiments, provided herein is are methods of delivering a protein into a plant, plant part, or plant cell the methods comprising contacting the plant with a composition comprising the protein, wherein the protein enters the plant and a) enters a plant cell, b) moves within the plant or plant part, or c) both (a) and (b). In some embodiments, the protein comprises a HypSys polypeptide, such as THSI (SEQ ID NO: 5) or THSII (SEQ ID NO: 6), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.

[0086] In some embodiments, provided herein are methods of delivering a protein into a plant, the methods comprising contacting the leaves of the plant with a composition comprising the protein, wherein the protein enters the plant and a) enters a plant cell, b) moves within the plant or plant part, or c) both (a) and (b). In some embodiments, the protein comprises a polypeptide selected from the group consisting of a HypSys polypeptide, CEP1 (SEQ ID NO: 1), and ELF18 (SEQ ID NO: 3), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.

[0087] In some embodiments, provided herein are methods of delivering a protein into a plant, the methods comprising spraying the leaves of the plant with a composition comprising the protein, the protein comprising a cargo domain and a polypeptide configured to enter the leaves of the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b). In some embodiments, the polypeptide comprises a plant signaling polypeptide, an elicitor polypeptide, or both.

[0088] In some embodiments, provided herein are methods of delivering a protein into a plant, plant part, or plant cell, the methods comprising contacting the plant, plant part, or plant cell with a composition comprising the protein, the protein comprising a cargo domain and a polypeptide configured to enter the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b), wherein the polypeptide comprises a plant signaling peptide, an elicitor polypeptide, or both.

[0089] In some embodiments, provided herein is are methods of delivering a protein into a plant, plant part, or plant cell, the methods comprising contacting the plant, plant part, or plant cell withPATENT APPLICATION DOCKET NO. P14953WOOOa composition comprising the protein, the protein comprising a cargo domain and a polypeptide configured to enter the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b). In some embodiments, the polypeptide comprises a plant signaling peptide, an elicitor polypeptide, or both from a source plant that is of a different family, genus, or species than the plant, plant part, or plant cell that is contacted with the composition. In other embodiments, the polypeptide is a cell-penetrating peptide, for example, pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity' thereto.

[0090] In some embodiments of the foregoing methods, the polypeptide is selected from the group consisting of a HypSy s polypeptide, a systemin polypeptide, pLAA (SEQ ID NO: 7), pLAAC (SEQ ID NO: 9), CpLAA (SEQ ID NO: 8), CEP1 (SEQ ID NO: 1), PEP1 (SEQ ID NO: 2), and ELF18 (SEQ ID NO: 3), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In certain embodiments, the HypSys polypeptide is a TobHypSys polypeptide, for example, THSI (SEQ ID NO: 5) or THSII (SEQ ID NO: 6), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity7thereto. In certain embodiments, the systemin polypeptide is tomato systemin (SEQ ID NO: 4), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity’ thereto.Methods of Foliar Application and Leaf Uptake

[0091] In some embodiments, provided herein are methods of delivering a protein into a plant, the methods comprising contacting the leaves of the plant with a composition comprising the protein, wherein the protein comprises a polypeptide configured to enter the leaves of the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b). In some embodiments, the protein further comprises a cargo domain that is covalently attached to the polypeptide or forms a complex with the polypeptide vianon-covalent interactions. In some embodiments, the contacting the leaves of the plant with the composition comprises applying one or more droplets of the composition onto the leaves of the plant. In some embodiments, the contacting the leaves of the plant with the composition comprises spraying the composition onto the leaves of the plant.

[0092] Methods of contacting leaves with compositions are known in the art and include methods suitable for laboratory, greenhouse, and field-scale applications. For example, contacting the leaves of the plant in a greenhouse or laboratory setting with a liquid composition may include may comprise spraying the leaves of the plant with the composition, manually applying one or morePATENT APPLICATION DOCKET NO. PI4953WOOOdroplets of the composition to the surface of the leaves, syringe or vacuum infiltration of the composition, incubation of the petiole of a cut leaf in the composition, and the like. In the setting of an agricultural field or a large-scale greenhouse setting, preferred methods for contacting the leaves of the plant include spraying and / or applying one or more droplets to the leaves of the plant, since methods such as syringe or vacuum infiltration or application to cut petioles are impractical in the context of crop production.

[0093] The contacting the leaves of the plant with the composition comprising the protein may be performed on any part of the leaf. For example, the composition may be applied to the surface of the leaf, such as by spraying or manual application of one or more droplets. In some embodiments, the methods comprise contacting (e.g., spraying) the surface of the leaf blade, the surface of the petiole, or both with the composition comprising the protein. In certain embodiments, the methods comprise contacting the surface of the leaf blade with the composition comprising the protein. In some embodiments, the methods comprise contacting (e.g., spraying) the adaxial surface of the leaf, the abaxial surface of the leaf, or both with the composition with the composition comprising the protein. In certain embodiments, the methods comprise contacting (e.g., spraying) the adaxial surface of the leaf with the composition comprising the protein. The adaxial surface of a leaf is the surface that faces toward the central axis (typically the central stem) of the plant. By contrast, the abaxial surface of a leaf is the surface that faces away from the central axis of the plant. For plants with leaf blades that are roughly horizontal, such as Arabidopsis or Palmer amaranth, the adaxial surface is generally the upper surface of the leaf, and the abaxial surface is generally the lower surface of the leaf.

[0094] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising spraying the leaves of the plant with a composition comprising the protein, wherein the protein enters the leaves of the plant and a) enters plant cells, b) moves within the plant, or c) both (a) and (b). In some embodiments, the polypeptide comprises a plant signaling polypeptide, an elicitor polypeptide, or both. In some embodiments, the protein further comprises a cargo domain that is covalently attached to the polypeptide or forms a complex with the polypeptide via non-covalent interactions.

[0095] Methods of spraying plant leaves are known in the art and include, for example, application of foliar sprays, aerial application, and the like. The spray may be applied using any means knownPATENT APPLICATION DOCKET NO. PI4953WOOOin the art, for example, a spray bottle, a handheld sprayer, a backpack sprayer, a boom sprayer, an airblast sprayer, an aerial sprayer, or a pull-type sprayer.

[0096] In some embodiments, the methods described herein comprise contacting leaves of a plant with the composition comprising the protein at ambient air pressure, wherein the protein enters the leaves of the plants and a) enters plant cells, b) moves within the plant, or c) both (a) and (b). In other words, in some embodiments, the contacting does not comprise syringe infiltration, vacuum infiltration, or any other form of infiltration that uses mechanical pressure. Ambient air pressure varies by location depending upon, for example, elevation. One of skill in the art will appreciate that ambient air pressure can range from 500 mbar to 1,100 mbar depending upon location, weather, and elevation. In certain embodiments, the contacting of the leaves comprises dropping or spraying one or more droplets of the composition comprising the protein above or near the adaxial or abaxial surface of the plant and allowing the one or more droplets to contact the leaf surface via gravity, wind, mechanical force, or any combination thereof.

[0097] In some embodiments of the methods described herein, the composition comprising the protein further comprises a surfactant. Without wishing to be bound by theory, co-application of the protein with a surfactant may promote even distribution of the protein on the plant surface and enhanced penetration into the plant. The composition may comprise any suitable surfactant known in the art or described herein.

[0098] In some embodiments of the methods described herein, the methods comprise pre-treating the leaves of the plant with a pretreatment composition comprising a surfactant prior to contacting or spraying the leaves of the plant with the composition comprising the protein. The methods of applying compositions to leaves that are known in the art or described herein may be used to apply the pre-treatment composition. For example, in some embodiments, applying the pre-treatment composition comprises contacting the leaves of the plant (e.g., the adaxial and / or abaxial surface of the leaf blade) with the pre-treatment composition. In some embodiments, the pre-treating comprises applying one or more droplets of the pre-treatment composition to one or more leaves of the plant prior to contacting the leaves with the composition comprising the protein. In certain embodiments, the pre-treating comprises spraying the leaves of the plant with the pretreatment composition prior to contacting the leaves (e.g., spraying the leaves) with the compositionPATENT APPLICATION DOCKET NO. P14953WOOOcomprising the protein. In some embodiments, the pre-treatment composition comprises a surfactant.

[0099] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising or consisting of a polypeptide selected from the group consisting of a HypSys polypeptide, a CEP1 polypeptide, or an ELF18 polypeptide, wherein the protein enters plant cells and moves within the plant. In some embodiments, the CEP polypeptide is CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In some embodiments, the ELF18 polypeptide comprises the amino acid sequence of SEQ ID NO: 3, or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity' thereto.

[0100] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising or consisting of a HypSys polypeptide, wherein the protein enters plant cells and moves within the plant. In some embodiments, the contacting comprises applying one or more droplets of the composition to the leaves of the plant. In certain embodiments, the contacting comprises spraying the composition onto the leaves of the plant. In some embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (III): X1ZX2X3ZX4X5, wherein each Z is P or O; XI is P, O, S, T, or A; X2 is P, O, S, or T; X3 is P, O, S, T, or A; X4 is K, T. A, or E; and X5 is P, O, S, T. H, K, or Y. In some embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (IV): X1OOX2OX3, wherein O is hydroxyproline; XI is P, O, S, or A; X2 is O, S, T, or A; and X3 is T, K, A, or E. In some embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (V): X1PPPX2PX3X4, wherein XI is P, L, or S; X2 is E, S, or A; X3 is K, Q, or E; and X4 is P, K, D. or H (SEQ ID NO:34). In certain embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (II): R(X1 )nX2ZX3X4ZX5X6X7X8X9Xl 0X11 XI 2, wherein n is 4, 5, or 6; each Z is P or O; Each XI is A, D, E, G, H, I, K, L, N, P, R, S, T, V, or Y; X2 is P, O, S, T, or A; X3 is P, O, S, or T; X4 is P, O, S. T, or A; X5 is K, T, A, or E; X6 is P, O, S, T, H, K, or Y; X7 is S. D, A. E, or Q; X8 is D, P, O, S, I, A, or no amino acid; X9 is P. O, G, E, I, H, S, or no amino acid; XI 0 is S, Q, T, I, Y, K, N, or no amino acid; XI 1 is K, R, H, N, G, or no amino acid; and XI 2 is Q, P, E, or no amino acid. In some embodiments, the HypSys polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6 and 16-PATENT APPLICATION DOCKET NO. P14953WOOO33, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In certain embodiments, the HypSys polypeptide is a TobHypSys polypeptide, e.g., THSI (SEQ ID NO:5) or THSII (SEQ ID NO:6). In some embodiments, the plant is a monocot (e.g., rye). In other embodiments, the plant is Palmer amaranth or Arabidopsis thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.

[0101] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising or consisting of a HypSys polypeptide, wherein the protein enters plant cells and moves within the plant, and wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (II): R(Xl)nX2ZX3X4ZX5X6X7X8X9X10Xl 1X12, wherein n is 4, 5, or 6; each Z is P or O; Each XI is A, D, E, G, H, I, K. L, N, P, R, S, T, V, or Y; X2 is P. O, 5, T, or A; X3 is P, O, S, or T: X4 is P, O, S, T. or A; X5 is K, T, A, or E; X6 is P. O, S. T, H. K, or Y; X7 is S, D, A, E, or Q; X8 is D, P, O, S, I, A, or no amino acid; X9 is P, O, G, E, I, H, S, or no amino acid; XI 0 is S, Q, T, I, Y, K, N, or no amino acid; XI 1 is K, R, H, N, G, or no amino acid; and X12 is Q, P, E, or no amino acid. In some embodiments, the contacting comprises applying one or more droplets of the composition to the leaves of the plant. In certain embodiments, the contacting comprises spraying the composition onto the leaves of the plant. In certain embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (IV): X1OOX2OX3, wherein O is hydroxyproline; XI is P, O, S, or A; X2 is O, S, T, or A; and X3 is T, K, A, or E. In some embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (V): X1PPPX2PX3X4, wherein XI is P, L, or S; X2 is E, S, or A; X3 is K, Q, or E; and X4 is P, K, D, or H (SEQ ID NO: 34). In certain embodiments, the HypSys polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6, 16-18, 22-27, and 31, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In certain embodiments, the HypSys polypeptide is a TobHypSys polypeptide, e.g., THSI (SEQ ID NO:5) or THSII (SEQ IDNO:6). In some embodiments, the plant is a monocot (e.g., rye). In other embodiments, the plant is Palmer amaranth or Arabidopsis thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.

[0102] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein,PATENT APPLICATION DOCKET NO. PI4953WOOOthe protein comprising or consisting of a HypSys polypeptide, wherein the protein enters plant cells and moves within the plant, and wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (IV): XI OOX2OX3, wherein O is hydroxyproline; XI is P, O, S, or A; X2 is O, S, T, or A; and X3 is T, K, A, or E. In some embodiments, the contacting comprises applying one or more droplets of the composition to the leaves of the plant. In certain embodiments, the contacting comprises spraying the composition onto the leaves of the plant. In certain embodiments, the HypSys polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6, 16-18, 22-27, and 31, or an amino acid sequence having at least 60%. 70%. 80%, 90%, or 95% sequence identity thereto. In certain embodiments, the HypSys polypeptide is aTobHypSys polypeptide, e.g., THSI (SEQ IDN0:5) or THSII (SEQ ID N0:6). In some embodiments, the plant is a monocot (e.g., rye). In other embodiments, the plant is Palmer amaranth or Arabidopsis thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.

[0103] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising or consisting of a TobHypSys polypeptide, wherein the protein enters plant cells and moves within the plant. In some embodiments, the TobHypSys polypeptide is THSI (SEQ ID NO: 5) or THSII (SEQ ID NO: 6), or a polypeptide having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity thereto. In certain embodiments, the TobHypSys polypeptide is THSII (SEQ ID NO: 6), or a polypeptide having at least 95% identity thereto. In some embodiments, the contacting comprises applying one or more droplets of the composition to the leaves of the plant. In some embodiments, the plant is a monocot (e.g., rye). In other embodiments, the plant is Palmer amaranth or Arabidopsis thaliana. In some embodiments, the composition comprises the protein in an amount of betw een 0.1 and 1.0 mg / mL.

[0104] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising or consisting of a THSII (SEQ ID NO: 6). or a polypeptide having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity thereto, wherein the protein enters plant cells and moves within the plant. In some embodiments, the protein comprises or consists of THSII (SEQ ID NO: 6). In some embodiments, the contacting comprises applying one or more droplets of the composition to the leaves of the plant. In some embodiments, the plant is aPATENT APPLICATION DOCKET NO. P14953WOOOmonocot (e.g., rye). In other embodiments, the plant is Palmer amaranth or Arabidopsis thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.

[0105] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising a systemin polypeptide and a cargo domain, wherein the protein enters plant cells and moves within the plant. In some embodiments, the contacting comprises applying one or more droplets of the composition to the leaves of the plant. In certain embodiments, the contacting comprises spraying the composition onto the leaves of the plant. In some embodiments, the systemin polypeptide comprises an amino acid sequence according to Formula (VI): PPX1KRX2PP, wherein XI is S or T, and X2 is D or P (SEQ ID NO: 35). In some embodiments, the systemin polypeptide comprises an amino acid sequence according to Formula (I): AX1X2SX3PPX4KRX5PPKMQTD, wherein XI is V or A; X2 is R, Q, or H; X3 is T or K; X4 is T or S; and X5 is D or P (SEQ ID NO: 15). In some embodiments, the systemin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 11-14, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In certain embodiments, the systemin polypeptide comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 95% identity thereto. In some embodiments, the plant is a monocot (e.g., rye). In other embodiments, the plant is Palmer amaranth or Arabidopsis lhaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.

[0106] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising a systemin polypeptide and a cargo domain, wherein the protein enters plant cells and moves within the plant, and wherein the systemin polypeptide comprises an amino acid sequence according to Formula (VI): PPX1KRX2PP, wherein XI is S or T, and X2 is D or P (SEQ ID NO: 35). In some embodiments, the systemin polypeptide comprises an amino acid sequence according to Formula (I): AX1X2SX3PPX4KRX5PPKMQTD, wherein XI is V or A; X2 is R, Q, or H; X3 is T or K; X4 is T or S: and X5 is D or P (SEQ ID NO: 15). In some embodiments, the systemin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 11-14, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95%PATENT APPLICATION DOCKET NO. P14953WOOOsequence identity thereto. In certain embodiments, the systemin polypeptide comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 95% identity thereto. In some embodiments, the plant is a monocot (e.g., rye). In other embodiments, the plant is Palmer amaranth or Arabidopsls thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.

[0107] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising a systemin polypeptide and a cargo domain, wherein the protein enters plant cells and moves within the plant, and wherein the systemin polypeptide comprises an amino acid sequence according to Formula (I): AX1X2SX3PPX4KRX5PPKMQTD, wherein XI is V or A; X2 is R, Q, or H; X3 is T or K; X4 is T or S; and X5 is D or P (SEQ ID NO: 15). In some embodiments, the systemin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 11-14, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In certain embodiments, the systemin polypeptide comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 95% identity thereto. In some embodiments, the plant is a monocot (e.g., rye). In other embodiments, the plant is Palmer amaranth or Arabidopsls thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.

[0108] In some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising a systemin polypeptide and a cargo domain, wherein the protein enters plant cells and moves within the plant, and wherein the systemin polypeptide the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 60%, 70%, 80%. 90%, or 95% sequence identity thereto. In certain embodiments, the systemin polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:4. In some embodiments, the plant is a monocot (e.g, ry e). In other embodiments, the plant is Palmer amaranth or Arabidopsls thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.

[0109] In some embodiments, provided herein is a method of delivering a protein into the leaves of a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising a cargo domain and pLAA (SEQ ID NO: 7), or a polypeptidePATENT APPLICATION DOCKET NO. PI4953WOOOhaving at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto, wherein the cargo domain and pLAA are covalently attached, and wherein the protein enters the plant and a) enters a plant cell, b) moves within the plant, or c) both (a) and (b). In some embodiments, the protein enters the plant cells and moves within the plant. In some embodiments, the protein comprises a peptide linker between the cargo domain and the pLAA. In some embodiments, the contacting comprises applying one or more droplets of the composition to the leaves of the plant. In certain embodiments, the contacting comprises spraying the composition onto the leaves of the plant. In some embodiments, the plant is a monocot (e.g, rye). In other embodiments, the plant is Palmer amaranth or Arabidopsis thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL. In some embodiments, the cargo domain and the pLAA are covalently attached via a peptide bond. In some embodiments, the cargo domain and the pLAA are covalently attached a triazole ring (e.g., via click chemistry).

[0110] In some embodiments, provided herein is a method of delivering a protein into the leaves of a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1); and pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA, wherein the CEP1 and the pLAA are fused via a peptide bond. In some embodiments, the protein comprises, fromN-terminusto C-terminus: CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1); and pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA. In other embodiments, the protein comprises, from N-terminus to C-terminus, pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA; and CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1). In certain embodiments, the protein comprises a peptide linker between the CEP1 and the pLAA. In some variations, the peptide linker comprises between 1 and 10 glycine and serine residues (e.g., G4S, SEQ ID NO: 10). In some embodiments, the protein comprises pLAAC (SEQ ID NO: 9), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAAC (SEQ ID NO: 9). In other embodiments, the protein comprises CpLAA (SEQ ID NO: 8), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CpLAA (SEQ ID NO: 8). In some embodiments, the contacting comprises applying one or morePATENT APPLICATION DOCKET NO. PI4953WOOOdroplets of the composition to the leaves of the plant. In certain embodiments, the contacting comprises spraying the composition onto the leaves of the plant. In some embodiments, the plant is a monocot (e.g., rye). In other embodiments, the plant is Palmer amaranth or Arabidopsis thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.[OHl] In some embodiments, provided herein is a method of delivering a protein into the leaves of a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising, from N-terminus to C-terminus, pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA; and CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1). In certain embodiments, the protein comprises a peptide linker between the CEP1 and the pLAA. In some variations, the peptide linker comprises between 1 and 10 glycine and serine residues (e.g., G4S, SEQ ID NO: 10). In Some embodiments, the protein comprises, from N-terminus to C-terminus, pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA; G4S (SEQ ID NO: 10); and CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1). In some embodiments, the protein comprises pLAAC (SEQ ID NO: 9), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAAC (SEQ ID NO: 9). In some embodiments, the contacting comprises applying one or more droplets of the composition to the leaves of the plant. In certain embodiments, the contacting comprises spraying the composition onto the leaves of the plant. In some embodiments, the plant is a monocot (e.g.. rye). In other embodiments, the plant is Palmer amaranth or Arabidopsis thaliana. In some embodiments, the composition comprises the protein in an amount of between 0.1 and 1.0 mg / mL.Methods of Soil Application and Root Uptake

[0112] In addition to spray application, the present invention also includes methods for delivering functional proteins to plants through soil application. In this method, the protein formulation is applied to the soil around the roots of the plant. The protein is taken up by the roots and moves systemically within the plant, reaching distant parts of the plant. This method provides an alternative approach to deliver functional proteins to plants, particularly for crops that are difficult to spray.PATENT APPLICATION DOCKET NO. PI4953WOOO

[0113] Accordingly, in some embodiments, provided herein is a method of delivering a protein into a plant, the method comprising contacting the roots of the plant with a composition comprising the protein, wherein the protein enters the roots of the plant and a) enters plant cells, b) moves within the plant, or c) both (a) and (b). In some embodiments, the polypeptide comprises a plant signaling polypeptide, an elicitor polypeptide, or both. In some embodiments, the protein further comprises a cargo domain that is covalently attached to the polypeptide or forms a complex with the polypeptide via non-covalent interactions.

[0114] In other embodiments, provided herein is a method of delivering a protein into a plant, the method comprising applying a composition comprising the protein to soil containing the plant, or a part thereof (e.g, roots, a seed, a cotyledon, or a sprout), wherein the protein enters the roots of the plant and a) enters plant cells, b) moves within the plant, or c) both (a) and (b). The composition may be applied to the soil in a liquid form (e.g.. by spraying a liquid) or a solid form (e.g.. by applying granules). In certain embodiments, the method comprises applying the composition to soil containing one or more seeds of the plant prior to emergence of the plant from the soil. In certain embodiments, the method comprises applying the composition to soil containing roots of intact plants (e.g., by side dressing). In some embodiments, the polypeptide comprises a plant signaling polypeptide, an elicitor polypeptide, or both. In some embodiments, the protein further comprises a cargo domain that is covalently attached to the polypeptide or forms a complex with the polypeptide via non-covalent interactions.

[0115] Methods of applying compositions to soil and plant parts (e.g., roots or seeds) therein are known in the art and include, for example, broadcasting prior to or at the time of sowing or planting, broadcasting after sowing or planting, in-furrow or drilling application at the time of sowing or planting, side dressing or top dressing after sowing, band placement prior to or at the time of sowing, injection into the soil, in irrigation water, and the like. Broadcasting includes broadcast spraying of liquid compositions and broadcast application of dry compositions (e.g., granules).Protein-Containing Compositions, Including Sprayable Protein Formulations

[0116] Also provided herein are formulations of compositions comprising the proteins. The formulations may be solid formulations or liquid formulations, for example, a sprayable liquid formulation. The spray able protein formulations of the present invention are designed to penetrate the plant cuticle, enter plant cells, and move systemically within the plant. These formulationsPATENT APPLICATION DOCKET NO. PI4953WOOOtypically include a functional protein, a surfactant to enhance penetration, and a suitable carrier. The functional protein can be fluorescently labeled with TAMRA to allow for visualization and tracking within the plant.

[0117] Accordingly, provided herein are compositions comprising the proteins described herein. In some embodiments, the composition comprising the protein is a liquid. In certain embodiments, the composition comprising the protein is a sprayable liquid. In some embodiments, the composition comprising the protein is a solid (e.g, a granule). In some embodiments, the composition comprises a suitable carrier for application to plants, plant parts, and / or plant cells. Suitable carriers include, for example, water, organic solvents, and other solvents used in agriculture and laboratory7practices. The compositions may also include any adjuvants (such as wetting agents), micronutrients, macronutrients, pesticides, biostimulants, microbial inoculants, plant hormones, and / or plant signaling agents including, for example, additional proteins.

[0118] In some embodiments, the compositions comprising the protein further comprise a surfactant. Surfactants are used in liquid formulations in agriculture as wetting and spreading agents when applying pesticides and other agricultural products to promote even distribution of the product and penetration into plants. In certain embodiments, the surfactant enhances penetration of epidermis. In certain embodiments, the surfactant enhances cuticle penetration. In some embodiments, the surfactant is a non-ionic surfactant, an anionic surfactant, a nitrogen-surfactant blend, an organosilicone, or an oil-surfactant blend. In certain embodiments, the surfactant is a non-ionic surfactant. The surfactant may be any surfactant known to be suitable for use in agricultural products. Non-ionic surfactants used in agriculture include, for example, polyethoxylated fatty7alcohols, alkyl polyglucosides, sorbitan, sorbitan esters, lauryl alcohol ethoxylates, acid ethoxylates, glyceryl esters, octylphenol ethoxylates, and ethanolamides. In certain embodiments the non-ionic surfactant is Silwet L-77®. Tween® 20, Tween® 80. Agral 90®, glycerol monolaurate, Silwet L-76®, ethoxylated nonylphenol (NP-9), morpholine, In-Zorb® 90, In-Zorb® ADV, Boost®, Activate Plus® adjuvant, and Kinetic®. Anionic surfactants used in agriculture include, for example, alkyl sulfates, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, sodium dodecyl sulfate, sodium alkyl ether sulfate, and sodium laureth sulfate. Nitrogen-surfactant blends used in agriculture can include any surfactant know n in the art or described herein in combination with a nitrogen source, for example, urea, anyhydrous ammonia, ammonium salts (e.g., ammonium nitrate or ammonium sulfate), or nitrate.PATENT APPLICATION DOCKET NO. P14953WOOO

[0119] Organosilicone surfactants used in agriculture include, for example, Silwet L-77®, Kinetic®, and silicone adjuvant D-10. Oil surfactant blends used in agriculture include, for example, Activator 90®, Agridex®, and Hasten®. These blends facilitate improved absorption and spreading of herbicides and other formulations.

[0120] In some embodiments, the compositions comprising the protein further comprise additional actives useful in agricultural applications. For example, in some embodiments, the composition comprising the protein further comprises a pesticide. The pesticide may comprise, for example, chlorpyrifos, lambda-cyhalothrin, imidacloprid, thiamethoxam, azoxystrobin, propi conazole, tebuconazole, fluopyram, cyfluthrin, spinosad, deltamethrin, acetamiprid, pyrethrin, chlorantraniliprole, emamectin benzoate, bromethalin, fipronil, methomyl, carbary l, thiodicarb, oxamyl, bifenthrin, metolachlor, flutolanil, dimethomorph, and / or other pesticides as described in the respective applications.

[0121] In some embodiments, the composition comprising the protein further comprises an herbicide. The herbicide may comprise, for example, ametryn, terbuthylazine, atrazine, alachlor, acetochlor, fomesafen. mesotrione, dicamba, nicosulfuron, rimsulfuron, hexazinone. metolachlor, S-metolachlor, glufosinate, glyphosate, clomazone, topramezone, imazethapyr, imazapic, sethoxydim, clethodim, thifensulfuron, sulfentrazone, pyrasulfotole, triclopyr, clopyralid, imazapyr, prometryn, pendimethalin, fluoroxypyr, picloram, chlorsulfuron, flumioxazin, oxadiazon, pyraflufen-ethyl. carfentrazone, saflufenacil, 2,4-dichlorophenoxyacetic acid, and / or other herbicides as described in the respective applications. The herbicide may have any site of action known in the art, including, for example, those shown in Table 1. In certain embodiments, the composition further comprises glyphosate, glufosinate, 2,4-dichlorophenoxyacetic acid, and / or dicamba.Table 1. Herbicidal sites of action and Herbicide Resistance Action Committee (HRAC) groups.PATENT APPLICATION DOCKET NO. P14953WOOO

[0122] In some embodiments, the composition is in liquid form and comprises the protein in an amount of at least about 0.01 mg / mL. In certain embodiments, the composition comprises the protein in an amount of at least about 0.1 mg / mL. In certain embodiments, the composition comprises the protein in an amount of at least about 0.25 mg / mL. In certain embodiments, the composition comprises the protein in an amount of at least about 0.50 mg / mL. In some embodiments, the composition comprises the protein in an amount of at least about 0.01 mg / mL, at least about 0.05 mg / mL, at least about 0.10 mg / mL, at least about 0.20 mg / mL, at least about 0.25 mg / mL, at least about 0.30 mg / mL, at least about 0.40 mg / mL, at least about 0.50 mg / mL, at least about 0.60 mg / mL, at least about 0.70 mg / mL, at least about 0.75 mg / mL, at least about 0.80 mg / mL, at least about 0.90 mg / mL, at least about 1.0 mg / mL, at least about 2.0 mg / mL, at leastPATENT APPLICATION DOCKET NO. P14953WOOOabout 3.0 mg / mL, at least about 4.0 mg / mL, at least about 5.0 mg / mL, at least about 6.0 mg / mL, at least about 7.0 mg / mL, at least about 8.0 mg / mL, at least about 9.0 mg / mL, or at least about 10 mg / mL.

[0123] In some embodiments, the composition is in liquid form and comprises the protein in an amount of between 0.01 mg / mL and 10 mg / mL. In certain embodiments, the composition comprises the protein in an amount of between 0.1 mg / mL and 1 mg / mL. In certain embodiments, the composition comprises the protein in an amount of between 0.01 mg / mL and 0.05 mg / mL, between 0.01 mg / mL and 0.1 mg / mL, between 0.01 mg / mL and 0.2 mg / mL, between 0.01 mg / mL and 0.3 mg / mL, between 0.01 mg / mL and 0.4 mg / mL, between 0.01 mg / mL and 0.5 mg / mL, between 0.01 mg / mL and 0.6 mg / mL, between 0.01 mg / mL and 0.7 mg / mL, between 0.01 mg / mL and 0.8 mg / mL, between 0.01 mg / mL and 0.9 mg / mL, between 0.01 mg / mL and 1.0 mg / mL, between 0.01 mg / mL and 2.5 mg / mL, between 0.01 mg / mL and 5.0 mg / mL, between 0.01 mg / mL and 10 mg / mL, between 0.05 mg / mL and 0.1 mg / mL, between 0.05 mg / mL and 0.2 mg / mL, between 0.05 mg / mL and 0.3 mg / mL, between 0.05 mg / mL and 0.4 mg / mL, between 0.05 mg / mL and 0.5 mg / mL, between 0.05 mg / mL and 0.6 mg / mL, between 0.05 mg / mL and 0.7 mg / mL, between 0.05 mg / mL and 0.8 mg / mL, between 0.05 mg / mL and 0.9 mg / mL, between 0.05 mg / mL and 1.0 mg / mL, between 0.05 mg / mL and 2.5 mg / mL, between 0.05 mg / mL and 5.0 mg / mL, between 0.05 mg / mL and 10 mg / mL, between 0.1 mg / mL and 0.2 mg / mL, between 0.1 mg / mL and 0.3 mg / mL, between 0.1 mg / mL and 0.4 mg / mL, between 0.1 mg / mL and 0.5 mg / mL, between 0.1 mg / mL and 0.6 mg / mL. between 0.1 mg / mL and 0.7 mg / mL, between 0.1 mg / mL and 0.8 mg / mL, between 0.1 mg / mL and 0.9 mg / mL, between 0.1 mg / mL and 1.0 mg / mL, between 0.1 mg / mL and 2.5 mg / mL, between 0.1 mg / mL and 5.0 mg / mL, between 0.1 mg / mL and 10 mg / mL, between 0.2 mg / mL and 0.3 mg / mL, between 0.2 mg / mL and 0.4 mg / mL, between 0.2 mg / mL and 0.5 mg / mL, between 0.2 mg / mL and 0.6 mg / mL, between 0.2 mg / mL and 0.7 mg / mL, between 0.2 mg / mL and 0.8 mg / mL, between 0.2 mg / mL and 0.9 mg / mL, between 0.2 mg / mL and 1.0 mg / mL, between 0.2 mg / mL and 2.5 mg / mL, between 0.2 mg / mL and 5.0 mg / mL, between 0.2 mg / mL and 10 mg / mL, between 0.3 mg / mL and 0.4 mg / mL, between 0.3 mg / mL and 0.5 mg / mL, between 0.3 mg / mL and 0.6 mg / mL, between 0.3 mg / mL and 0.7 mg / mL, between 0.3 mg / mL and 0.8 mg / mL, between 0.3 mg / mL and 0.9 mg / mL, between 0.3 mg / mL and 1.0 mg / mL. between 0.3 mg / mL and 2.5 mg / mL, between 0.3 mg / mL and 5.0 mg / mL, between 0.3 mg / mL and 10 mg / mL, between 0.4 mg / mL and 0.5 mg / mL, between 0.4 mg / mL and 0.6 mg / mL, between 0.4 mg / mL and 0.7 mg / mL,PATENT APPLICATION DOCKET NO. P14953WOOObetween 0.4 mg / mL and 0.8 mg / mL, between 0.4 mg / mL and 0.9 mg / mL, between 0.4 mg / mL and 1.0 mg / mL, between 0.4 mg / mL and 2.5 mg / mL, between 0.4 mg / mL and 5.0 mg / mL, between 0.4 mg / mL and 10 mg / mL, between 0.5 mg / mL and 0.6 mg / mL, between 0.5 mg / mL and 0.7 mg / mL, between 0.5 mg / mL and 0.8 mg / mL, between 0.5 mg / mL and 0.9 mg / mL. between 0.5 mg / mL and 1.0 mg / mL, between 0.5 mg / mL and 2.5 mg / mL, between 0.5 mg / mL and 5.0 mg / mL, between 0.5 mg / mL and 10 mg / mL, between 0.6 mg / mL and 0.7 mg / mL, between 0.6 mg / mL and 0.8 mg / mL, between 0.6 mg / mL and 0.9 mg / mL, between 0.6 mg / mL and 1.0 mg / mL, between 0.6 mg / mL and 2.5 mg / mL, between 0.6 mg / mL and 5.0 mg / mL, between 0.6 mg / mL and 10 mg / mL, between 0.7 mg / mL and 0.8 mg / mL, between 0.7 mg / mL and 0.9 mg / mL, between 0.7 mg / mL and 1.0 mg / mL, between 0.7 mg / mL and 2.5 mg / mL, between 0.7 mg / mL and 5.0 mg / mL, between 0.7 mg / mL and 10 mg / mL, between 0.8 mg / mL and 0.9 mg / mL, between 0.8 mg / mL and 1.0 mg / mL, between 0.8 mg / mL and 2.5 mg / mL, between 0.8 mg / mL and 5.0 mg / mL, between 0.8 mg / mL and 10 mg / mL, between 0.9 mg / mL and 1.0 mg / mL. between 0.9 mg / mL and 2.5 mg / mL. between 0.9 mg / mL and 5.0 mg / mL, between 0.9 mg / mL and 10 mg / mL, between 1.0 mg / mL and 2.5 mg / mL, between 1.0 mg / mL and 5.0 mg / mL, between 1.0 mg / mL and 10 mg / mL, between 2.5 mg / mL and 5.0 mg / mL, between 2.5 mg / mL and 10 mg / mL, or between 5.0 mg / mL and 10 mg / mL.

[0124] In some embodiments, the composition is in liquid form and comprises the protein in an amount of about 0.01 mg / mL, about 0.05 mg / mL, about 0.10 mg / mL, about 0.20 mg / mL, about 0.25 mg / mL, about 0.30 mg / mL, about 0.40 mg / mL, about 0.50 mg / mL, about 0.60 mg / mL, about 0.70 mg / mL, about 0.75 mg / mL, about 0.80 mg / mL, about 0.90 mg / mL, about 1.0 mg / mL, about 2.0 mg / mL, about 3.0 mg / mL, about 4.0 mg / mL, about 5.0 mg / mL, about 6.0 mg / mL, about 7.0 mg / mL, about 8.0 mg / mL, about 9.0 mg / mL, or about 10 mg / mL. In certain embodiments, the composition comprises the protein in an amount of about 1.0 mg / mL. In certain embodiments, the composition comprises the protein in an amount of about 0.5 mg / mL. In certain embodiments, the composition comprises the protein in an amount of about 0.25 mg / mL. In certain embodiments, the composition comprises the protein in an amount of about 0.10 mg / mL.

[0125] In some embodiments, the composition is in liquid form, the protein comprises a cargo domain and a polypeptide configured to a) enter plant cells, b) move within the plant, or c) both (a) and (b), and comprises the polypeptide in an amount of at least about 0.01 mg / mL. In certain embodiments, the composition comprises the polypeptide in an amount of at least about 0.1 mg / mL. In certain embodiments, the composition comprises the polypeptide in an amount of atPATENT APPLICATION DOCKET NO. P14953WOOOleast about 0.25 mg / rnL. In certain embodiments, the composition comprises the polypeptide in an amount of at least about 0.50 mg / mL. In some embodiments, the composition comprises the polypeptide in an amount of at least about 0.01 mg / mL, at least about 0.05 mg / mL, at least about 0.10 mg / mL, at least about 0.20 mg / mL, at least about 0.25 mg / mL, at least about 0.30 mg / mL, at least about 0.40 mg / mL, at least about 0.50 mg / mL, at least about 0.60 mg / mL, at least about 0.70 mg / mL, at least about 0.75 mg / mL, at least about 0.80 mg / mL, at least about 0.90 mg / mL, at least about 1.0 mg / mL, at least about 2.0 mg / rnL, at least about 3.0 mg / mL, at least about 4.0 mg / mL, at least about 5.0 mg / mL, at least about 6.0 mg / mL, at least about 7.0 mg / mL, at least about 8.0 mg / mL, at least about 9.0 mg / mL. or at least about 10 mg / mL.

[0126] In some embodiments, the composition is in liquid form, the protein comprises a cargo domain and a polypeptide configured to a) enter plant cells, b) move within the plant, or c) both (a) and (b). and comprises the polypeptide in an amount of between 0.01 mg / mL and 10 mg / mL. In certain embodiments, the composition comprises the polypeptide in an amount of between 0.1 mg / mL and 1 mg / mL. In certain embodiments, the composition comprises the polypeptide in an amount of between 0.01 mg / mL and 0.05 mg / mL, between 0.01 mg / mL and 0.1 mg / mL, between 0.01 mg / mL and 0.2 mg / mL, between 0.01 mg / mL and 0.3 mg / mL, between 0.01 mg / rnL and 0.4 mg / mL, between 0.01 mg / mL and 0.5 mg / mL, between 0.01 mg / mL and 0.6 mg / mL, between 0.01 mg / mL and 0.7 mg / mL, between 0.01 mg / mL and 0.8 mg / rnL, between 0.01 mg / mL and 0.9 mg / mL, between 0.01 mg / mL and 1.0 mg / mL, between 0.01 mg / mL and 2.5 mg / mL, between 0.01 mg / mL and 5.0 mg / rnL, between 0.01 mg / mL and 10 mg / mL, between 0.05 mg / mL and 0.1 mg / mL, between 0.05 mg / mL and 0.2 mg / rnL, between 0.05 mg / mL and 0.3 mg / mL, between 0.05 mg / mL and 0.4 mg / mL, between 0.05 mg / mL and 0.5 mg / rnL, between 0.05 mg / mL and 0.6 mg / mL, between 0.05 mg / mL and 0.7 mg / mL, between 0.05 mg / mL and 0.8 mg / mL, between 0.05 mg / mL and 0.9 mg / mL, between 0.05 mg / mL and 1.0 mg / rnL, between 0.05 mg / mL and 2.5 mg / mL, between 0.05 mg / mL and 5.0 mg / rnL, between 0.05 mg / mL and 10 mg / mL. between 0.1 mg / mL and 0.2 mg / mL, between 0.1 mg / mL and 0.3 mg / mL, between 0.1 mg / rnL and 0.4 mg / mL, between 0.1 mg / mL and 0.5 mg / mL, between 0.1 mg / mL and 0.6 mg / mL, between 0.1 mg / mL and 0.7 mg / rnL, between 0.1 mg / mL and 0.8 mg / rnL, between 0.1 mg / mL and 0.9 mg / mL, between 0.1 mg / mL and 1.0 mg / mL, between 0.1 mg / mL and 2.5 mg / mL, between 0.1 mg / mL and 5.0 mg / mL, between 0.1 mg / mL and 10 mg / mL, between 0.2 mg / mL and 0.3 mg / mL, between 0.2 mg / mL and 0.4 mg / rnL, between 0.2 mg / mL and 0.5 mg / rnL, between 0.2 mg / mL and 0.6 mg / mL, between 0.2PATENT APPLICATION DOCKET NO. P14953WOOOmg / mL and 0.7 mg / mL, between 0.2 mg / mL and 0.8 mg / mL, between 0.2 mg / mL and 0.9 mg / mL, between 0.2 mg / mL and 1.0 mg / mL, between 0.2 mg / mL and 2.5 mg / mL, between 0.2 mg / mL and 5.0 mg / mL, between 0.2 mg / mL and 10 mg / mL, between 0.3 mg / mL and 0.4 mg / mL, between 0.3 mg / mL and 0.5 mg / mL, between 0.3 mg / mL and 0.6 mg / mL, between 0.3 mg / mL and 0.7 mg / mL, between 0.3 mg / mL and 0.8 mg / mL, between 0.3 mg / mL and 0.9 mg / mL, between 0.3 mg / mL and 1.0 mg / mL, between 0.3 mg / mL and 2.5 mg / mL, between 0.3 mg / mL and 5.0 mg / mL, between 0.3 mg / mL and 10 mg / mL, between 0.4 mg / mL and 0.5 mg / mL, between 0.4 mg / mL and 0.6 mg / mL, between 0.4 mg / mL and 0.7 mg / mL, between 0.4 mg / mL and 0.8 mg / mL, between 0.4 mg / mL and 0.9 mg / mL, between 0.4 mg / mL and 1.0 mg / mL, between 0.4 mg / mL and 2.5 mg / mL, between 0.4 mg / mL and 5.0 mg / mL, between 0.4 mg / mL and 10 mg / mL, between 0.5 mg / mL and 0.6 mg / mL, between 0.5 mg / mL and 0.7 mg / mL, between 0.5 mg / mL and 0.8 mg / mL, between 0.5 mg / mL and 0.9 mg / mL, between 0.5 mg / mL and 1.0 mg / mL, between 0.5 mg / mL and 2.5 mg / mL, between 0.5 mg / mL and 5.0 mg / mL, between 0.5 mg / mL and 10 mg / mL, between 0.6 mg / mL and 0.7 mg / mL, between 0.6 mg / mL and 0.8 mg / mL, between 0.6 mg / mL and 0.9 mg / mL, between 0.6 mg / mL and 1.0 mg / mL, between 0.6 mg / mL and 2.5 mg / mL, between 0.6 mg / mL and 5.0 mg / mL, between 0.6 mg / mL and 10 mg / mL, between 0.7 mg / mL and 0.8 mg / mL, between 0.7 mg / mL and 0.9 mg / mL, between 0.7 mg / mL and 1.0 mg / mL, between 0.7 mg / mL and 2.5 mg / mL, between 0.7 mg / mL and 5.0 mg / mL, between 0.7 mg / mL and 10 mg / mL, between 0.8 mg / mL and 0.9 mg / mL, between 0.8 mg / mL and 1.0 mg / mL, between 0.8 mg / mL and 2.5 mg / mL, between 0.8 mg / mL and 5.0 mg / mL, between 0.8 mg / mL and 10 mg / mL, between 0.9 mg / mL and 1.0 mg / mL, between 0.9 mg / mL and 2.5 mg / mL, between 0.9 mg / mL and 5.0 mg / mL, between 0.9 mg / mL and 10 mg / mL, between 1.0 mg / mL and 2.5 mg / mL, between 1.0 mg / mL and 5.0 mg / mL, between 1.0 mg / mL and 10 mg / mL, between 2.5 mg / mL and 5.0 mg / mL, between 2.5 mg / mL and 10 mg / mL, or between 5.0 mg / mL and 10 mg / mL.

[0127] In some embodiments, the composition is in liquid form, the protein comprises a cargo domain and a polypeptide configured to a) enter plant cells, b) move within the plant, or c) both (a) and (b), and comprises the polypeptide in an amount of about 0.01 mg / mL, about 0.05 mg / mL, about 0.10 mg / mL, about 0.20 mg / mL, about 0.25 mg / mL, about 0.30 mg / mL, about 0.40 mg / mL, about 0.50 mg / mL. about 0.60 mg / mL, about 0.70 mg / mL, about 0.75 mg / mL, about 0.80 mg / mL, about 0.90 mg / mL, about 1.0 mg / mL, about 2.0 mg / mL, about 3.0 mg / mL, about 4.0 mg / mL, about 5.0 mg / mL, about 6.0 mg / mL, about 7.0 mg / mL, about 8.0 mg / mL, about 9.0 mg / mL, or about 10PATENT APPLICATION DOCKET NO. P14953WOOOmg / mL. In certain embodiments, the composition comprises the protein in an amount of about 1.0 mg / mL. In certain embodiments, the composition comprises the protein in an amount of about 0.5 mg / mL. In certain embodiments, the composition comprises the protein in an amount of about 0.25 mg / mL. In certain embodiments, the composition comprises the protein in an amount of about 0.10 mg / mL.

[0128] In some embodiments, the composition is a solid formulation, such as granules or a powder. In certain embodiments, the formulation is activated to allow for protein delivery’ to the plant, for example, by wetting on the plant or soil by natural processes (e.g, precipitation or condensation) or agricultural (e.g., irrigation, watering) processes.Protein Penetration and Systemic Movement

[0129] The ability of the sprayable protein formulations to penetrate the plant cuticle and move systemically within the plant is a key aspect of the invention. This is achieved by, for example, applying the protein formulation to the leaves of an intact plant using a fine mist sprayer. The protein penetrates the cuticle, enters the plant cells, and moves systemically to distant parts of the plant, such as leaves and stems. This systemic movement is essential for delivering the functional protein to various parts of the plant, thereby enhancing grow th and resistance to diseases.

[0130] Plant biology and anatomy presents many barriers to the entry of substances into the plant, particularly the arial portions of the plant. In normal life processes the areal portions of the plant exchange gasses (like CO2 and 02, carbon dioxide and oxygen) with the atmosphere, through living, controlled pores on the lower (abaxial) leaf surface. These pores, called stomata, are opened and closed by the plants in response to needs for gas exchange, and to regulate loss of water vapor, which occurs through the stomata as w ell. The rest of the leaf surface is covered with a waxy protective layer, called the cuticle, which is composed of complex fatty acid derived waxes, which largely prevent the movement of water and w ater vapor through the leaf surfaces. This waxy barrier must also be transited by any crop protection chemicals that are intended to enter the cell. All plant cells also contain a cell wall, surrounding each of the cells, composed of complex carbohydrates. This barrier must also be transited before an applied molecule can access the outer surface of the plant cell membrane. Finally the membrane itself must be transited to gain access to the cell.PATENT APPLICATION DOCKET NO. PI4953WOOO

[0131] The loss of water vapor from the stomata creates tension on the water movement system of the plant, the xylem, and generally causes the mass flow of water from the roots to the shoots of the plant. This moving water stream is how necessary mineral nutrients enter the plant through the roots and are distributed throughout the aerial portions of the plant for use. This movement stream can also move other water-soluble molecules in a generally upward direction and is sometimes used by the plant to move mobile signals that control growth and development.

[0132] An alternative downward based movement system, called the phloem, is used by the plant in the alternate direction (typically downward, from shoot to root). This system is driven by osmotically generated pressure flow. High solute concentrations (typically sugars) in the shoot, which are produced in photosynthesis are created in the cells of the phloem in the leaf, this high solute concentration pulls in water, which creates the pressure that drives downward movement of solutions in the phloem system.

[0133] For an applied molecule to fully access all parts of the plant it must be moved in one or both of these systems, after making entry through the cuticle, through the cell wall and through the cell membrane.

[0134] The state of the art shows cell entry7of certain peptides when applied to intact leaves, but almost always with additional mechanical force (vacuum infiltration or "needless syringe' pressure application). The instant invention, in some embodiments, gains entry without mechanical forces and provides systemic movement when motifs of cell penetrating peptides (CPP)s are combined with known endogenous systemic signaling peptides (some of which move, and some of which do not, when applied in this way).

[0135] In some embodiments of the methods described herein, the protein enters plant tissue, for example, by penetrating the surface of plant tissue and entering the sub-surface portion of the plant tissue. In certain embodiments, the protein enters plant leaves, plant roots, or both. In some embodiments, the protein enters plant tissues by penetrating the plant epidermis (e.g., the leaf epidermis or the root epidermis), or a part thereof. In some embodiments, the protein enters plant leaves by penetrating the leaf cuticle. The leaf cuticle may be penetrated, for example, by entry through cuticle pores, or by entry7through the stomata into the xylem.PATENT APPLICATION DOCKET NO. PI4953WOOO

[0136] In some embodiments of the methods described herein, the protein enters plant cells. The protein may enter plant cells by, for example, directly penetrating the cuticle through cuticle pores and entering epidermal cells; penetrating the cuticle through stomata, entering the xylem, and penetrating cells from within the xylem: penetrating and entering guard cells (i.e., the cells found at the opening of stomata) on the abaxial or adaxial leaf surface; and the like.

[0137] In some embodiments, the protein enters plant cells. In certain embodiments, the protein enters epidermal cells. In certain embodiments, the protein enters mesophyll cells (e.g. palisade cells and / or spongy mesophyll cells). In certain embodiments, the protein enters vascular cells. In certain embodiments, the protein enters guard cells. In certain embodiments, the protein enters parenchymal cells. In certain embodiments, the protein enters collenchyma cells. In certain embodiments, the protein enters sclerenchyma cells.

[0138] In some embodiments, the protein enters one or more organelles within the plant cells. In certain embodiments, the protein enters the nucleus. In certain embodiments, the protein enters the chloroplast. In certain embodiments, the protein enters the mitochondria. In certain embodiments, the protein enters the vacuole. In some embodiments, the protein enters the endoplasmic reticulum, Golgi apparatus, and / or peroxisomes.

[0139] In some embodiments of the methods described herein, after entering plant tissue, the protein moves within the plant or plant tissue. In some embodiments, protein moves within the xylem of the plant or plant tissue (e.g. , leaf). In certain embodiments, the protein moves within the xylem of the plant or plant tissue (e.g., leaf) in an acropetal direction (e.g., toward the meristem or leaf tip, ty pically upward). In some embodiments, protein moves within the phloem of the plant or plant tissue (e.g. leaf). In certain embodiments, the protein moves within the phloem of the plant or plant tissue (e.g. , leaf) in a basipetal direction (e.g., toward the base of the stem or roots, typically downward). In some embodiments, the protein moves within both the xylem and the phloem of the plant or plant tissue (e.g., leaf). In certain embodiments, the protein moves within both the xylem in an acropetal direction and the phloem in a basipetal direction. The protein may also move within the plant or plant tissue via the apoplast, the symplast, or both. The apoplast is the non-living component of plant tissue consisting of intercellular spaces bordered by cell walls. The symplast is a living component of plant tissue, comprising the interconnected cytoplasms of cells connected by channels in the cell walls called plasmodesmata.PATENT APPLICATION DOCKET NO. P14953WOOOPlant Type and Species

[0140] The proteins and compositions described herein may be applied to any kind or species of plant. For example, the plant may be a food or industrial crop plant, an ornamental plant, a home garden plant, a grass, a medicinal plant, a landrace, a pest plant (weed), and the like. In some embodiments, the plant is an intact plant. In some embodiments, the plant is present in an agricultural setting, e.g., in a field, greenhouse, irrigation system, or hydroponic system. In some embodiments, the plant is present in a lawn or garden.

[0141] In some embodiments, the plant is a food or industrial crop, such as, for example, maize, rye, alfalfa, apple, artichoke, asparagus, avocado, banana, barley, basil, beans (e.g., soybeans, common beans, and the like), beets, berries, blackberry’, blueberry, broccoli, brussels sprouts, cabbage, cacao, carrot, cashew, cassava, cauliflower, celery, chard, chickpea, chives, citrus (e.g., orange, lemon, lime, grapefruit, citron, and the like), clover, coffee, collards, cotton, cranberry, cucumber, currant, eggplant, fig, flax, garlic, ginger, grape, guava, hazelnut, hemp, hop, kale, kiwi, kohlrabi, leek, lentil, lettuce, maple, melon, millet, miscanthus, mustard, oat, oil palms, okra, olive, onion, papaya, parsnip, peanut, pea, pear, penny cress, pepper (e.g., sweet peppers, hot peppers, and the like), peppercorn, pine, pineapple, pistachio, pomegranate, poplar, potato, quinoa, radish, rapeseed, raspberry, rice, rose, safflower seed, scallion, sesame, shallot, sorghum, soybean, spinach, squash, stone fruit (e.g., cherry', peach, plum, apricot, nectarine, almond, and the like), strawberry, sunflower, sugarcane, sweet potato, switchgrass, tea, teff, tobacco, tomatillo, tomato, turf grass, turnip, walnut, wheat, yams, or yucca.

[0142] In some embodiments, the plant is a weed, such as, for example, Palmer amaranth (Amaranthus palmeri), common lambsquarters (Chenopodium album), waterhemp (Amaranthus tuberculatus), horseweed / marestail (Conyza canadensis), morning glory species (Ipomoea spp.), yellow nutsedge (Cyperus esculentus), purple nutsedge (Cyperus rotundus), kochia (Bassia scoparia), common ragweed (Ambrosia artemisiifolia), giant ragweed (Ambrosia trifida), eastern black nightshade (Solanum ptychanthum), hairy nightshade (Solarium physalifolium), giant foxtail (Setaria faberi), green and yellow foxtail (Setaria viridis, Setaria pumila), giant foxtail (Setaria faberi), bamyardgrass (Echinochloa crus-galli), Johnsongrass (Sorghum halepense), Italian ryegrass (Lolium miiltiflorum), brome species (Bromus spp.), Canada thistle (Cirsium arvense), horsenettle (Solanum carolinense), annual bluegrass (Boa annua), crabgrass species (DigitariaPATENT APPLICATION DOCKET NO. PI4953WOOOspp.), velvetleaf (Abutilon theophrasti), wild oat (Avena fatua), redroot pigweed (Amaranthus retroflexus), witchgrass (Panicum capillar e), fall panicum Panicum dichotomiflorum), johnsongrass (Sorghum hale pens e), quackgrass Elymus repens'), nutsedge (Cyperus spp ), or bermudagrass (Cynodon dactylori).

[0143] In some embodiments, the plant is a monocot. Monocot plants include, for example, plants in the family Poaceae. Accordingly, in some embodiments, the plant is a plant of the family Poaceae. In some embodiments, the plant of the family Poaceae is a cultivated crop or ornamental plant, for example, maize (Zea mays), rye (Secale cereale), barley (Hordeum vulgare), oats (Avena sativa), rice (Oryza sativa or Oryza glaberrima), sorghum (Sorghum bicolor), wheat (Triticum spp., e.g., Triticum aestivum), millet (subfamilies Panicoideae and Chloridoideae), sugarcane (Saccharum officinarum), lawn and ornamental grasses (for example, Poa spp., Agrostis spp., Festuca spp., Calamagrostis spp., Deschampsia spp., Lolium spp., bamboo, and the like), Brachypodium spp. (e.g., Brachypodium distachyon), and the like. In other embodiments, the plant is a weed species in the family Poaceae, for example, annual bluegrass (e.g., Poa annua), bamyardgrass (e.g., Echinochloa crus-galli), cogon grass (Imperata cylindrical), crabgrass (Digitaria spp.). foxtail (Setaria spp.), quackgrass (Elymus repens), ryegrass (Lolium spp.), sandbur (Cenchrus spp.), velvet grass (Holcus lanatus), and the like. In certain embodiments of any of the methods described herein, the plant is rye or maize.

[0144] In some embodiments, the plant is a dicot. Dicot plants include, for example, plants of the families Brassicaceae and Amaranthaceae. Accordingly, in some embodiments, the plant is a plant of the family Brassicaceae. In some embodiments, the plant of the family Brassicaceae is a cultivated crop or ornamental plant, for example, crops of the species Brassica oleraceae (e.g., broccoli, Brussels sprouts, cabbage, cauliflower, collard greens, kale, kohlrabi, and Savoy cabbage), crops of the species Brassica rapa (e.g.. bok choy, mizuna. napa cabbage, rapini, and turnip), arugula (Eruca sativa), garden cress (Lepidium sativum), horseradish (Armoracia rusticana), mustard (white mustard, Sinapis alba; brown mustard, Brassica j uncea; black mustard, B. nigra), radish (Raphanus), rapeseed (Brassical napus), wasabi (Eutrema japonicum), watercress (Nasturtium officinale). In some embodiments, the plant of the family Brassicaceae is a model organism, for example, Arabidopsis spp. (e.g, Arabidopsis thaliana). In other embodiments, the plant of the family Brassicaceae is a weed, for example, field pennycress (Thlaspi arvense), field pepperweed (Lepidium campestre), garlic mustard (AlliariaPATENT APPLICATION DOCKET NO. PI4953WOOOpetiolata), hairy bittercress (Cardamine hirsuta), marsh yellowcress (Rorippa pai'uslris). shepherd's purse (Capsella bursa-pastoris), Virginia pepperweed (Lepidium virginicum), wild mustard (Brassica kaber), wild radish (Raphanus raphanistrum), or yellow rocket (Barbarea vulgaris). In certain embodiments of any of the methods described herein, the plant is Arabidopsis thaliana.

[0145] In other embodiments, the plant is a plant of the family Amaranthaceae. In some embodiments, the plant of the family Amaranthaceae is a cultivated crop, a medicinal plant, or an ornamental plant, for example, Alternanthera spp., Amaranthus spp., Beta vulgaris (beet, chart), Celosia spp., Dysphania anthelmintica, epazote (Dysphania ambrosioides), Iresine spp., kaniwa (Chenopodium pallidi caiile), lamb's quarters (Chenopodium berlandieri), quinoa (Chenopodium quinoa), Salicornia spp., spinach (Spinacia oleracea). In other embodiments, the plant of the family Amaranthaceae is a weed or invasive species, for example, alligatorweed (Alternanthera philoxeroides), Bassia scoparia, common water hemp (Amaranthus nidus), Palmer amaranth (Amaranthus palmeri), redroot pigweed (Amaranthus retroflexus), roughfruit amaranth (Amaranthus tuberculatus), Salsola tragus, or smooth amaranth (Amaranthus hybridus). In certain embodiments of any of the methods described herein, the plant is Palmer amaranth.

[0146] In some embodiments of the methods described herein, the protein, or a part thereof, is derived from a source organism of a different family, genus, and / or species than the plant, plant part, or plant cell that is contacted with the composition comprising the protein. For example, the cargo domain and / or the polypeptide configured to enter the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b) are from a source organism that is of a different family, genus, and / or species than the plant, plant part, or plant cell that is contacted with the composition comprising the protein. In some embodiments, the source organism is a bacterium (the source organism for ELF 18). In other embodiments, the source organism is a plant of a different family, genus, and / or species than the plant, plant part, or plant cell that is contacted with the composition comprising the protein. In certain embodiments, the source organism is a dicot plant, and the plant, plant part, or plant cell that is contacted with the composition comprising the protein is a monocot plant, plant part, or plant cell. In some embodiments, the source organism is tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), ox Arabidopsis thaliana. In certain embodiments, the source organism is tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), ox Arabidopsis thaliana, and the plant, plant part, or plant cell that is contacted with the composition comprisingPATENT APPLICATION DOCKET NO. P14953WOOOthe protein is of a species other than tobacco (Nicotiana tabacum), tomato (Solarium lycopersicum), or Arabidosis thaliana.

[0147] In some embodiments, the protein or a part thereof (e.g.. the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the order Solanales (also referred to as Solanaceous origin proteins). In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the order Solanales, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is of an order other than Solanales. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the order Solanales, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a monocot. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the order Solanales, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the order Poales (e.g., ry e or maize). In certain embodiments, the protein or a part thereof (e.g. , the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the order Solanales, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the order Brassicales (e.g., Arabidopsis thaliana). In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the order Solanales, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the order Caryophyllales (e.g.. Palmer amaranth). In some variations, the protein or a part thereof (e.g., the polypeptide configured to enter the plant) from the plant of the order Solanales comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 11-14, and 16-33, or an amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity thereto. In certain embodiments, the plant of the order Solanales is a plant of the family Solanaceae. In some variations, the protein or a part thereof (e.g, the polypeptide configured to enter the plant) from the plant of the family Solanaceae comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 11-14, and 16-27, or an amino acid sequences having at least 60%, at least 70%. at least 80%, at least 90%, or at least 95% identity thereto. In other embodiments, the plant of the order Solanales is a plant of the family Convolvulaceae (e.g., Ipomoea batatas). In some variations, the protein or a part thereof (e.g. , the polypeptide configuredPATENT APPLICATION DOCKET NO. P14953WOOOto enter the plant) from the plant of the family Convolvulaceae comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, or an amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity thereto.

[0148] In some embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Solanaceae. Plants of the family Solanaceae include, for example, plants of the genera Solarium, Nicotiana, Capsicum, and Petunia. In certain embodiments, the protein or a part thereof (e.g, the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Solanaceae, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is of a family other than Solanaceae. In certain embodiments, the protein or a part thereof (e.g, the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Solanaceae, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a monocot. In certain embodiments, the protein or a part thereof (e.g, the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Solanaceae, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the family Poaceae (e.g. rye or maize). In certain embodiments, the protein or a part thereof (e.g, the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Solanaceae, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the family Brassicaceae (e.g, Arabidopsis thaliana). In certain embodiments, the protein or a part thereof (e.g, the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Solanaceae, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the family Amaranthaceae (e.g, Palmer amaranth). In some variations, the protein or a part thereof (e.g, the polypeptide configured to enter the plant) from the plant of the family Solanaceae comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6. 11-14, and 16-27, or an amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity thereto. In some variations, the protein or a part thereof (e.g., the polypeptide configured to enter the plant) from the plant of the family Solanaceae comprises systemin (SEQ ID NO: 4), THSI (SEQ ID NO: 5). THSII (SEQ ID NO: 6), or a polypeptide having an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical thereto.PATENT APPLICATION DOCKET NO. PI4953WOOO

[0149] In some embodiments, the protein or a part thereof {e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the genus Solanum {e.g., Solanum lycopersicum), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of a genus other than Solanum. Plants of the genus Solanum include, for example, S. lycopersicum. S. tuberosum, and S. nigrum. In certain embodiments, the protein or a part thereof {e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the genus Solanum e.g., Solanum lycopersicum), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a monocot. In certain embodiments, the protein or a part thereof {e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the genus Solanum {e.g., Solanum lycopersicum), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the genus Zea {e.g. , maize), Secale {e.g. , rye), Arabidopsis {e.g. , Arabidopsis thaliana), or Amaranthus {e.g. Palmer amaranth). In some variations, the protein or a part thereof {e.g., the polypeptide configured to enter the plant) from the plant of the genus Solanum comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 11-13, 16-21, and 25-27, or an amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity thereto. In some variations, the protein or a part thereof {e.g, the polypeptide configured to enter the plant) from the plant of the genus Solanum is systemin (SEQ ID NO: 4) or a polypeptide having an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical thereto.

[0150] In some embodiments, the protein or a part thereof {e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from Solanum lycopersicum (tomato), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of a species other than Solanum lycopersicum. In certain embodiments, the protein or a part thereof {e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from Solanum lycopersicum, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a monocot. In certain embodiments, the protein or a part thereof {e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from Solanum lycopersicum, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is maize, rye. Arabidopsis thaliana or Palmer amaranth. In some variations, the protein or a part thereof (e.g., the polypeptide configured to enter the plant) from Solanum lycopersicum is systeminPATENT APPLICATION DOCKET NO. PI4953WOOO(SEQ ID NO: 4) or a polypeptide having an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical thereto.

[0151] In some embodiments, the protein or a part thereof (e.g.. the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the genus Nicotianum (e.g. , Nicotianum tabacum), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of a genus other than Nicotianum. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the genus Nicotianum (e.g. , Nicotianum tabacum), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a monocot. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the genus Nicotianum (e.g., Nicotianum tabacum), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the genus Zea (e.g., maize), Secale (e.g., rye). Arabidopsis (e.g., Arabidopsis thaliana), or Amaranthus (e.g., Palmer amaranth). In some variations, the protein or a part thereof (e.g. , the polypeptide configured to enter the plant) from the plant of the genus Nicotianum is THSI (SEQ ID NO: 5), THSII (SEQ ID NO: 6). or a polypeptide having an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical thereto.

[0152] In some embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from Nicotianum tabacum (tobacco), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of a species other than Nicotianum tabacum. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from Nicotianum tabacum, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a monocot. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from Nicotianum tabacum, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is maize, rye, Arabidopsis thaliana or Palmer amaranth.. In some variations, the protein or a part thereof (e.g., the polypeptide configured to enter the plant) from Nicotianum tabacum is THSI (SEQ ID NO: 5), THSII (SEQ ID NO: 6), or a polypeptide having an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical thereto.PATENT APPLICATION DOCKET NO. PI4953WOOO

[0153] In some embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Brassicaceae. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Brassicaceae, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is of a family other than Brassicaceae. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Brassicaceae, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a monocot. In certain embodiments, the protein or a part thereof (e.g, the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Brassicaceae, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the family Poaceae (e.g., rye or maize). In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the family Brassicaceae, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the family Amaranthaceae (e.g., Palmer amaranth). In some variations, the protein or a part thereof (e.g., the polypeptide configured to enter the plant) from the plant of the family Brassicaceae is CEP1 (SEQ ID NO: 1), PEP (SEQ ID NO: 2), or a polypeptide having an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical thereto.

[0154] In some embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the genus Arabidopsis (e.g., Arabidopsis thaliana), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of a genus other than Arabidopsis. In certain embodiments, the protein or a part thereof (e.g.. the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the genus Arabidopsis (e.g.. Arabidopsis thaliana), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a monocot. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from a plant of the genus Arabidopsis (e.g. , Arabidopsis thaliana), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of the genus Zea (e.g. , maize). Secale (e.g. , rye), or Amaranthus (e.g. , Palmer amaranth). In some variations, the protein or a part thereof (e.g., the polypeptide configured to enter the plant)PATENT APPLICATION DOCKET NO. PI4953WOOOfrom the plant of the genus Arabidopsis is CEP1 (SEQ ID NO: 1), PEP (SEQ ID NO: 2), or a polypeptide having an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical thereto.

[0155] In some embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from Arabidopsis thaliana (also referred to as Arabidopsis origin peptides), and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a plant of a species other than Arabidopsis thaliana. In certain embodiments, the protein or a part thereof (e.g, the cargo domain and / or the polypeptide configured to enter the plant) is from Arabidopsis thaliana, and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is a monocot. In certain embodiments, the protein or a part thereof (e.g., the cargo domain and / or the polypeptide configured to enter the plant) is from Arabidopsis thaliana. and the plant, plant part, or plant cell that is contacted by the composition comprising the protein is maize, rye, or Palmer amaranth. In some variations, the protein or a part thereof (e.g., the polypeptide configured to enter the plant) from Arabidopsis thaliana is CEP1 (SEQ ID NO: 1), PEP (SEQ ID NO: 2), or a polypeptide having an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical thereto.Methods for Discovering and Developing Functional Proteins

[0156] The present invention also includes methods for discovering and developing functional proteins that can penetrate the plant cuticle, enter plant cells, and move systemically within the plant. One such method is the intact plant TAMRA assay. In this assay, proteins are fluorescently labeled with TAMRA and applied to the leaves of an intact plant. Fluorescent and brightfield images are recorded at multiple time points after application to monitor the penetration, entry, and systemic movement of the protein within the plant. This assay allows for the identification and characterization of proteins that are effective in penetrating the plant cuticle and moving systemically within the plant.

[0157] Other exemplary methods for discovering and developing functional proteins that enter plant cells and / or move within plants include protein binding assays. For example, a protein or a part thereof (e.g, a cargo domain) may be detected within plant tissue by using an antibody-based assay that specifically recognizes the protein (or cargo domain). This would allow application of the protein to an intact plant at an application site, sampling of tissue that is either basipetally orPATENT APPLICATION DOCKET NO. PI4953WOOOacropetally distant from the application site, and detection of the protein in the tissue sample. Any type of protein binding assay may be used, for example, Western blot, ELISA, and the like. Similarly, a nucleic acid cargo domain may be detected using a nucleic acid probe designed to specifically bind to the cargo domain (e.g, a complementary nucleic acid).Experimental Peptides and Proteins

[0158] The present invention includes a variety of experimental peptides and proteins that have been tested for their ability to penetrate the plant cuticle, enter plant cells, and move systemically within the plant. These include Arabidopsis origin signal peptides (e.g., AtNSl, also referred to as CEP or CEP1; Pepl), Solanaceous origin signal peptides (e.g., Systemin, TobHypSys2, TobHypSysl), bacterial origin elicitor peptides (ELF18), and plant demonstrated CPPs in culture and with injection (e.g, pLysAibALA (pLAA)). Combinations of these peptides, such as Cep-G4S-pLAA and pLAA-G4S-Cep, have also been tested and shown to be effective.

[0159] Biopesticides, particularly insecticidal proteins, have established themselves as important crop protection solutions, particularly when deployed in transgenic plants (GMOs) as plant incorporated protectants (PIPs). The origins of these PIPs have been in natural organisms such as Bacillus thuringensis (Bt) and these proteins have been further developed and optimized using the tools of molecular genetics to provide even higher levels of control, and to control new organisms. More recently, a small number of these proteins have been approved and commercialized as spray on controls.

[0160] To be successful in crop protection and crop enhancement novel biopesticides and other bioactive agents need to be functional when applied to the plant through conventional spray systems. This means, that when applied in an aqueous solution, the applied biochemical must be absorbed into plant cells and / or mobilized throughout the plant so they can reach portions of the plant that are not contacted by the initial spray process.

[0161] Proteins, seeds, plants, and parts thereof produced by methods described herein are also part of the disclosure.

[0162] The present invention provides a comprehensive approach to delivering functional proteins into plants, offering significant advantages over traditional methods. The sprayable and soilPATENT APPLICATION DOCKET NO. P14953WOOOapplication methods, along with the discovery and development assays, provide a versatile and efficient solution for enhancing plant grow th, resistance to diseases, and overall productivity.

[0163] Accordingly, in some embodiments, provided herein are isolated and / or recombinant proteins configured to enter plant cells, move within plants, or both. In some embodiments, the polypeptide is configured to enter the leaves of the plant by entering through stomata, by penetrating the cuticle, or both. In some embodiments, the polypeptide is configured to enter plant cells. In some embodiments, the polypeptide is configured to move within the plant. In some embodiments, the polypeptide is configured to both enter plant cells and to move within the plant. In some embodiments, the polypeptide comprises or consists of a plant signaling or elicitor polypeptide. In some embodiments, the isolated and / or recombinant protein further comprises a cargo domain. In certain embodiments, the cargo domain is covalently attached to the polypeptide. The cargo domain and the polypeptide may be covalently attached using any means known in the art. For example, in some embodiments, the isolated and / or recombinant protein comprises the cargo domain and the polypeptide fused via a peptide bond. In other embodiments, the cargo domain and polypeptide are covalently attached via a conjugation method. In certain embodiments, the cargo domain and the polypeptide are covalently attached via click chemistry, to form a conjugate with a triazole ring connecting the cargo domain and the polypeptide. In other embodiments, the cargo domain forms a complex with the polypeptide via non-covalent interactions (e.g, ionic interaction). Sequences of exemplary' polypeptides configured to enter plant cells, move within the plant, or both are provided in Table 2.Table 2. Exemplary polypeptides configured to enter plant cells, move within the plant, or both, and associated sequences. “O’" represents hydroxyproline, “B” represents 2-aminoisobutync acid.PATENT APPLICATION DOCKET NO. P14953WOOO<<<PATENT APPLICATION DOCKET NO. P14953WOOOPATENT APPLICATION DOCKET NO. P14953WOOOPATENT APPLICATION DOCKET NO. P14953WOOO* Pearce, G. (2011). Systemin, hydroxyproline-rich systemin and the induction of protease inhibitors. Current Protein and Peptide Science, 12(5), 399-408.

[0164] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both is between 5 and 40 amino acids in length. In certain embodiments, the polypeptide is between 10 and 40 amino acids in length. In certain embodiments, the polypeptide is between 15PATENT APPLICATION DOCKET NO. P14953WOOOand 35 amino acids in length. In certain embodiments, the is a plant signaling polypeptide or an elicitor polypeptide between 15 and 22 amino acids in length. In certain embodiments, the polypeptide configured to enter plant cells, move within plants, or both is between 5 and 10, between 5 and 15. between 5 and 20, between 5 and 25, between 5 and 30, between 5 and 35, between 5 and 40, between 10 and 15, between 10 and 20, between 10 and 25, between 10 and 30, between 10 and 35, between 10 and 40, between 15 and 20, between 15 and 25, between 15 and 30, between 15 and 35, between 15 and 40, between 20 and 25, between 20 and 30, between 20 and 35. between 20 and 40, between 25 and 30. between 25 and 35, between 25 and 40. between 30 and 35, between 30 and 40. or between 35 and 40 amino acids in length.

[0165] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both is around 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in length. In certain embodiments, the polypeptide is around 18 amino acids in length.

[0166] In some embodiments, at least 2 of the amino acid residues in the polypeptide configured to enter plant cells, move within plants, or both are basic amino acid residues. In some embodiments, at least 2 of the residues in the polypeptide configured to enter plant cells, move within plants, or both are lysine, arginine, and / or histidine residues. In certain embodiments, at least 2, 3, 4, 5, 6, 7 or 8 of the residues of the polypeptide are lysine, arginine, and / or histidine residues. In certain embodiments, between 2 and 3, between 2 and 4, between 2 and 5, between 2 and 6, between 2 and 7, between 2 and 8, between 3 and 4, between 3 and 5, between 3 and 6, between 3 and 7, between 3 and 8, between 4 and 5, between 4 and 6, between 4 and 7, between 4 and 8, between 5 and 6, between 5 and 7, between 5 and 8, between 6 and 7, between 6 and 8, or between 7 and 8 of the residues of the polypeptide are lysine, arginine, and / or histidine residues. In certain embodiments, between 3 and 5 of the residues of the polypeptide are lysine, arginine, and / or histidine residues. In certain embodiments, around 4 of the residues of the polypeptide are lysine, arginine, and / or histidine residues.

[0167] In some embodiments, at least 10% of the amino acid residues in the polypeptide configured to enter plant cells, move within plants, or both are basic amino acid residues. In some embodiments, at least 10% of the amino acid residues in the polypeptide configured to enter plant cells, move within plants, or both are lysine, arginine, and / or histidine residues. In certain embodiments, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%PATENT APPLICATION DOCKET NO. P14953WOOOof the amino acid residues in the polypeptide are lysine, arginine, and / or histidine residues. In some embodiments, between 10% and 40% of the amino acids in the polypeptide configured to enter plant cells, move within plants, or both are lysine, arginine, and / or histidine residues. In certain embodiments, between 20% and 30% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues. In certain embodiments, between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25% and 30%, between 25% and 35%, or between 30% and 35% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues.

[0168] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid residues in the polypeptide configured to enter plant cells, move within plants, or both are proline and / or hydroxyproline residues. In certain embodiments, at least 3 of the amino acid residues in the polypeptide are proline and / or hydroxyproline residues. In certain embodiments, at least 4 of the amino acid residues in the polypeptide are proline and / or hydroxy proline residues. In some embodiments, between 1 and 8 of the amino acid residues in the polypeptide configured to enter plant cells, move within plants, or both are proline and / or hydroxyproline residues. In certain embodiments, between 1 and 2, between 1 and 3, between 1 and 4, between 1 and 5, between 1 and 6, between 1 and 7, between 1 and 8, between 2 and 3, between 2 and 4, between 2 and 5, between 2 and 6, between 2 and 7, between 2 and 8, between 3 and 4. between 3 and 5, between 3 and 6, between 3 and 7, between 3 and 8, between 4 and 5, between 4 and 6, between 4 and 7, between 4 and 8, between 5 and 6, between 5 and 7, between 5 and 8, between 6 and 7, between 6 and 8, or between 7 and 8 of the amino acid residues in the polypeptide are proline and / or hydroxyproline residues.

[0169] In some embodiments, between 15% and 50% of the amino acid residues in the polypeptide configured to enter plant cells, move within plants, or both are proline and / or hydroxyproline residues. In certain embodiments, between 20% and 45% of the amino acid residues in the polypeptide are proline and / or hydroxy proline residues. In certain embodiments, between 30% and 35% of the amino acid residues in the polypeptide are proline and / or hydroxyproline residues. In certain embodiments, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%,PATENT APPLICATION DOCKET NO. P14953WOOObetween 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%. between 35% and 40%, between 35% and 45%, between 35% and 50%, between 40% and 45%, between 40% and 50%, or between 45% and 50% of the amino acid residues in the polypeptide are proline and / or hydroxyproline residues.

[0170] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both comprises one or more hydroxyproline (O) residues. In some embodiments, at least 1, 2, 3, 4, 5, or 6 of the amino acid residues in the polypeptide configured to enter plant cells, move within plants, or both are hydroxy proline residues. In certain embodiments, at least 3 of the amino acid residues in the polypeptide are hydroxyproline residues. In some embodiments, between 1 and 6 of the amino acid residues in the polypeptide configured to enter plant cells, move within plants, or both are hydroxyproline residues. In certain embodiments, between 1 and 2, between 1 and 3, between 1 and 4, between 1 and 5, between 1 and 6, between 2 and 3, between 2 and 4, between 2 and 5, between 2 and 6, between 3 and 4, between 3 and 5, between 3 and 6, between 4 and 5, between 4 and 6, or between 5 and 6 of the amino acid residues in the polypeptide are hydroxyproline residues.

[0171] In some embodiments, between 15% and 35% of the amino acid residues in the polypeptide configured to enter plant cells, move within plants, or both are hydroxyproline residues. In certain embodiments, between 15% and 20% of the amino acid residues in the polypeptide are hydroxy proline residues. In certain embodiments, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25% and 30%, between 25% and 35%, or between 30% and 35% of the amino acid residues in the polypeptide are hydroxyproline residues.

[0172] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both lacks one or more post-translational modification relative to a native polypeptide. For example, in some embodiments, the polypeptide lacks one or more carbohydrate (e.g., pentose) moieties relative to a native polypeptide. In certain embodiments, the polypeptide lacks all carbohydrate (e.g., pentose) moieties relative to a native polypeptide. In some embodiments, thePATENT APPLICATION DOCKET NO. P14953WOOOpolypeptide is unglycosylated. In some embodiments, the polypeptide lacks one or more proline hydroxylations that are present in a native polypeptide. In certain embodiments, the polypeptide lacks all proline hydroxylations that are present in a native polypeptide. In certain embodiments, the polypeptide is unhydroxylated. In some variations of the foregoing embodiments, the polypeptide and the native polypeptide have primary amino acid sequences (i.e., prior to post-translational modification, e.g., prior to glycosylation and / or hydroxylation) that are at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to one another. In some embodiments, the polypeptide is a variant of one or more polypeptides described herein, in which one or more or each of the hydroxyproline (O) residues is substituted with a proline (P) residue. In certain variations, each of the hydroxyproline (O) residues is substituted with a proline (P) residue.

[0173] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both comprises or consists of a plant signaling polypeptide or an elicitor polypeptide. Plant signaling polypeptides include any polypeptide that is involved in a signal transduction pathway within plants. The signaling pathway may be involved in defense against pathogens and herbivores, response to abiotic stress, and the like. Plant elicitor polypeptides play a role in eliciting an immune response in plants, and include, for example, microbe-associated molecular patterns (MAMPs), pathogen-associated molecular patterns (PAMPs), and damage-associated molecular patterns (DAMPs). Plant signaling and elicitor polypeptides are described extensively in the art and reviewed, for example, in Jones and Dangl 2006 (The plant immune system. Nature 444, 323-329), Boiler and Felix 2009 (A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual review of plant biology, 60, 379-406), Pearce 2011 (Systemin, hydroxyproline-rich systemin and the induction of protease inhibitors. Current Protein and Peptide Science, 12(5), 399-408), Chen et al 2019 (The role of peptides cleaved from protein precursors in eliciting plant stress reactions. New Phygologist, 225(6). 2267-2282). Zhang et al. 2020 (Systemin-mediated long-distance systemic defense responses. New Phytologist, 226(6), 1573-1582), and Zelman and Berkowitz 2023 (Plant Elicitor Peptide (Pep) Signaling and Pathogen Defense in Tomato. Plants (Basel). Aug 3;12(15):2856. doi: 10.3390 / plantsl2152856. PMID: 37571010; PMCID: PMC10421127). Exemplary plant signaling peptides include, for example, systemin polypeptides (e.g. SEQ ID NOs: 4 and 11-14), HypSys polypeptides (e.g., SEQ ID NOs: 5-6 and 16-33), CEP polypeptides (e.g., CEPL SEQ ID NO: 1), PEP polypeptides (e.g., PEP1, SEQ ID NO: 2), and homologous polypeptides, e.g., polypeptidesPATENT APPLICATION DOCKET NO. P14953WOOOhaving at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity to a systemin polypeptide (e.g, SEQ ID NOs: 4 or 11-14), a HypSys polypeptide (e.g, SEQ ID NOs: 5-6 or 16-33), a CEP polypeptide (e.g., CEP1, SEQ ID NO: 1), a PEP polypeptide (e.g. PEP1, SEQ ID NO: 2). Elicitor polypeptides include, for example, ELF18 (SEQ ID NO: 3), and homologous polypeptides, e.g, polypeptides having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity' to ELF18 (SEQ ID NO: 3).Systemin Polypeptides

[0174] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both is a systemin polypeptide. Systemin polypeptides are signaling polypeptides that are involved in plant defense responses to pathogens and herbivores. Systemin polypeptides have been identified in plants of the genus Solanum, including tomato (SEQ ID NO: 4), potato (SEQ ID NOs: 11-12), S. nigrum (SEQ ID NO: 13), and pepper (SEQ ID NO: 14) and are characterized by containing the sequence PPX1KRX2PP, wherein XI is S or T and X2 is D or P (SEQ ID NO: 35). This is considered a palindromic sequence, in that two central basic amino acids (KR) are flanked by pairs of prolines (PP). Systemin peptides are derived from a precursor protein called prosystemin, which is processed post-translationally to yield the 18-amino-acid systemin peptide. Systemin polypeptides have been shown to move within plants in a root-to-shoot direction (Mucha et al. 2019 "Capillary electrophoresis study of systemin peptides spreading in tomato plant." Electrophoresis 40.2: 336-342.). Systemin polypeptides are reviewed, for example, in Ryan and Pearce 2003 (Systemins: a functionally defined family of peptide signals that regulate defensive genes in Solanaceae species. PNAS, 100(suppl_2), 14577-14580), Pearce 2011 (Systemin, hydroxyproline-rich systemin and the induction of protease inhibitors. Current Protein and Peptide Science, 12(5), 399-408) and Zhang et al. 2020 (Systemin-mediated long-distance systemic defense responses. New Phytologist, 226(6), 1573-1582). Tomato systemin peptides have been applied to tomato plants for the purpose of priming plant defenses and increasing resistance to pests (Coppola et al. 2019. Tomato plants treated with systemin peptide show enhanced levels of direct and indirect defense associated with increased expression of defense-related genes. Plants, 8(10), 395). However, there are no reports of using systemin polypeptides as a means of delivering a cargo domain into a plant, and no reports of applying systemin from one plant to a plant of a different species.PATENT APPLICATION DOCKET NO. P14953WOOO

[0175] In some embodiments, the systemin polypeptide comprises an amino acid sequence according to Formula (VI) PPX1KRX2PP, wherein XI is S or T and X2 is D or P (SEQ ID NO: 35). In some embodiments, the systemin polypeptide comprises an amino acid sequence according to Formula (I): AX1X2SX3PPX4KRX5PPKMQTD, wherein XI is V or A; X2 is R. Q, or H; X3 is T or K; X4 is T or S; and X5 or D or P (SEQ ID NO: 15). In some embodiments, the systemin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 11-14, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In some embodiments, the systemin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 11-14. or a variant thereof comprising up to 7 (e.g., up to 1, 2, 3, 4, 5, 6, or 7) amino acid substitutions. In certain embodiments, the systemin polypeptide comprises the amino acid sequence of SEQ ID NO: 4. HypSys Polypeptides

[0176] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both is a HypSys polypeptide. HypSys polypeptides are signal polypeptides originating from plants within the order Solanales. The first HypSys polypeptides. TobHypSysI (THSI. SEQ ID NO: 5) and TobHypSysII (THSII, SEQ ID NO: 6), were discovered in tobacco (Pearce et al. 2001. Production of multiple plant hormones from a single polyprotein precursor. Nature, 411(6839), 817-820; THSI and THSII renumbered in Pearce 2011. Systemin, hydroxyproline-rich systemin and the induction of protease inhibitors. Current Protein and Peptide Science, 12(5), 399-408), followed by TomHypSysI (SEQ ID NO: 16), TomHypSysII (SEQ ID NO: 17), and TomHypSysIII (SEQ ID NO: 18) isolated from tomato (Pearce and Ryan 2003. Systemic signaling in tomato plants for defense against herbivores: isolation and characterization of three novel defense-signaling glycopeptide hormones coded in a single precursor gene. Journal of Biological Chemistry. 278(32), 30044-30050). In the name “HypSys."’ “Hyp” refers to the presence of hydroxyproline residues in THSI and THSII, and “Sys” refers to the hypothesis that THSI and THSII were systemin-like signal peptides. Like systemin, HypSys signal polypeptides are derived from a larger precursor protein that is post-translationally processed to yield HypSys polypeptides. THSI and THSII are further post-translationally modified through glycosylation and hydroxylation of proline residues to yield hydroxyproline residues. Several HypSys polypeptides that are both hydroxylated and glycosylated have been discovered (SEQ ID NOs: 5-6, 16-18, 22-27, and 31), all of which contain a central hydroxyproline-rich domain according to Formula (IV): XI OOX2OX3,PATENT APPLICATION DOCKET NO. P14953WOOOwherein O is hydroxyproline; XI is P, O, S, or A; X2 is O, S, T, or A; and X3 is T, K, A, or E. In addition, several HypSys polypeptides have been discovered to have similar primary sequences while hydroxylation and glycosylation (SEQ ID NOS: 19-21, 28-30, and 32-33), most of which contain a central proline-rich domain according to Formula (V): X1PPPX2PX3X4, wherein XI is P, L, or S; X2 is E, S, or A; X3 is K, Q, or E; and X4 is P, K, D, or H (SEQ ID NO: 34). HypSys polypeptides are reviewed, for example, in Ryan and Pearce 2003 (Systemins: a functionally defined family of peptide signals that regulate defensive genes in Solanaceae species. PNAS, 100(suppl_2), 14577-14580), Pearce 2011, and Zhang et al. 2020 (Systemin-mediated longdistance systemic defense responses. New Phytologist, 226(6). 1573-1582). There are no reports of applying HypSys polypeptides to intact plants, using them as a means of delivering a cargo domain into a plant, or applying a HypSys polypeptide from one plant to a plant of a different species.

[0177] In some embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (III): X1ZX2X3ZX4X5, wherein each Z is P or O; XI is P, O, S, T, or A; X2 is P, O, S, or T; X3 is P, O, S, T, or A; X4 is K, T, A, or E; and X5 is P, O, S, T, H, K, or Y, wherein O is hydroxyproline. In some embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (IV): XI OOX2OX3, wherein O is hydroxyproline; XI is P, O, S, or A; X2 is O, S, T, or A; and X3 is T, K, A, or E. In some embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (V): X1PPPX2PX3X4, wherein XI is P. L, or S; X2 is E, S, or A; X3 is K, Q, or E; and X4 is P, K, D, or H (SEQ ID NO:34). In some embodiments, the HypSys polypeptide comprises an amino acid sequence according to Formula (II): R(Xl)nX2ZX3X4ZX5X6X7X8X9X10Xl 1X12, wherein n is 4, 5, or 6; each Z is P or O; Each XI is A, D, E, G, H, I, K, L, N, P, R, S, T, V, or Y; X2 is P, O, S, T, or A; X3 is P, O, S, or T; X4 is P, O, S, T, or A; X5 is K, T, A, or E; X6 is P. O, S, T, H, K, or Y; X7 is S, D, A, E. or Q; X8 is D, P, O. S, I, A. or no amino acid; X9 is P, O, G, E, I. H, S. or no amino acid; X10 is S, Q, T, I, Y, K, N, or no amino acid; XI 1 is K, R, H, N, G, or no amino acid; and XI 2 is Q, P, E, or no amino acid, wherein O is hydroxyproline. In some embodiments, the HypSys polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6 and 16-33, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In some embodiments, the HypSys polypeptide is a TobHypSys polypeptide. In certain embodiments, the TobHypSys polypeptide is THSI (SEQ ID NO: 5) or THSII (SEQ IDPATENT APPLICATION DOCKET NO. P14953WOOONO: 6), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In some embodiments, the HypSys polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6 and 16-33, or a variant thereof comprising up to 8 (e.g., up to 1, 2. 3, 4. 5, 6. 7, or 8) amino acid substitutions. In certain embodiments, the HypSys polypeptide comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the HypSys polypeptide comprises the amino acid sequence of SEQ ID NO: 5.

[0178] In some embodiments, the HypSys polypeptide lacks one or more post-translational modification relative to a native HypSys polypeptide. For example, in some embodiments, the HypSys polypeptide lacks one or more pentose moieties relative to a native HypSys polypeptide. In certain embodiments, the HypSys polypeptide lacks all pentose moieties relative to a native HypSys polypeptide. In some embodiments, the HypSys polypeptide is unglycosylated. In some embodiments, the HypSys polypeptide lacks one or more proline hydroxylations that are present in a native HypSys polypeptide. In certain embodiments, the HypSys polypeptide lacks all proline hydroxylations that are present in a native HypSys polypeptide. In certain embodiments, the HypSys polypeptide is unhydroxylated. In some variations of the foregoing embodiments, the HypSys polypeptide and the native HypSys polypeptide have primary amino acid sequences (i.e., prior to post-translational modification, e.g, prior to glycosylation and / or hydroxylation) that are at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to one another. In some embodiments, the HypSys polypeptide is a variant of one or more HypSys polypeptides described herein, in which one or more or each of the hydroxy proline (O) residues is substituted with a proline (P) residue. In certain variations, each of the hydroxyproline (O) residues is substituted with a proline (P) residue.CEP Polypeptides

[0179] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both is a CEP polypeptide (e.g., a CEP1 polypeptide), also known as C -TERMINALLY ENCODED PEPTIDE (CEP). CEP polypeptides are signaling peptides involved in plant response to abiotic stresses (Ohyama et al. 2008. Identification of a biologically active, small, secreted peptide in Arabidopsis by in silico gene screening, followed by LC-MS-based structure analysis. The Plant Journal, 55(1), 152-160). CEP1, first identified in Arabidopsis thaliana, was found to be a root-to-shoot, xylem-mobile signal involved in the plant response to low nitrogen availabilityPATENT APPLICATION DOCKET NO. PI4953WOOO(Ohkubo et al. 2017. Shoot-to-root mobile polypeptides involved in systemic regulation of nitrogen acquisition. Nature plants, 3(4), 1-6). Similar to other plant signaling peptides described herein, CEP polypeptides are also derived from a precursor polypeptide. In the case of CEP poly peptides, the precursor contains one or more CEP domains, as well as an N-terminal secretion signal, a variable domain, and a C-terminal extension, which are processed to yield the 15 amino acid CEP peptides. Hundreds of CEP polypeptides have been discovered in both angiosperms and gymnosperms, and CEP domains are described in detail in Ogilvie et al. 2014 (Diversification of the C-TERMINALLY ENCODED PEPTIDE (CEP) gene family in angiosperms, and evolution of plant-family specific CEP genes. BMC genomics, 15(1 ). 870). CEP polypeptides have been applied to the roots of plants in multiple studies to elucidate the role of the CEP polypeptides in various signaling pathways (e.g., Tabata et al. 2014. Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling. Science, 346(6207), 343-346; Laffont et al. 2020. The NIN transcription factor coordinates CEP and CLE signaling peptides that regulate nodulation antagonistically. Nature communications, 11(1), 3167; Roy et al. 2022. Application of synthetic peptide CEP1 increases nutrient uptake rates along plant roots. Frontiers in Plant Science, 12, 793145). However, there are no reports of using CEP polypeptides, such as CEP1, as a means of delivering a cargo domain into a plant.

[0180] In some embodiments, the CEP polypeptide is aCEPl polypeptide. In some embodiments, the CEP polypeptide is an Arabidopsis thaliana CEP1 polypeptide. In some embodiments, the CEP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 1. or an amino acid sequence having at least 60%, 70%, 80%, 90%. or 95% sequence identity thereto. In some embodiments, the CEP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 6 (e.g., up to 1, 2, 3, 4, 5, or 6) amino acid substitutions. In certain embodiments, the CEP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 1.PEP Polypeptides

[0181] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both is a PEP polypeptide (e.g. , a PEP1 polypeptide). Plant elicitor peptides (PEPs) are signaling peptides involved in response to plant pests. Plants have been shown to express PEPs in response to several stressors, including pathogens, herbivores, wounding, and plant hormone treatments. Similar to other plant signaling peptides described herein, PEP polypeptides are also derived fromPATENT APPLICATION DOCKET NO. P14953WOOOa precursor polypeptide called PROPEP, which is processed post-translationally to yield one or more PEPs about 20-23 amino acids in length (Huffaker and Ryan 2007. Endogenous peptide defense signals in Arabidopsis differentially amplify signaling for the innate immune response. Proceedings of the National Academy of Sciences, 104(25), 10732-10736). A wide variety ofPEP polypeptides have been discovered across many plant families, including Brassicaceae (e.g., Arabidopsis thaliana), Rosaceae, Poaceae, Solanaceae, and Fabaceae (Ruiz et al. 2018. Novel Rosaceae plant elicitor peptides as sustainable tools to control Xanthomonas arboricola pv. pruni in Prunus spp. Molecular Plant Pathology, 19(2). 418-431; Zelman et al. 2024. Pathogen elicitor peptide (pep), systemin, and their receptors in tomato: sequence analysis sheds light on standing disagreements about biotic stress signaling components. BMC Plant Biol 24, 7280). PEP polypeptides from A. thaliana, maize (Zea mays'), and peach (Primus persica) have been sprayed onto their respective source plants in order to study PEP signaling mechanisms and prime plant defenses against pathogens and other stressors (Y amaguchi et al. 2010. PEPR2 is a second receptor for the Pepl and Pep2 peptides and contributes to defense responses in Arabidopsis. The Plant Cell, 22(2), 508-522; Huffaker et al. 2011 ZmPepl, an ortholog of Arabidopsis elicitor peptide 1, regulates maize innate immunity and enhances disease resistance. Plant physiology, 155(3), 1325- 1338; Foix et al. 2021. Prunus persica plant endogenous peptides PpPepl and PpPep2 cause PTI-like transcriptome reprogramming in peach and enhance resistance to Xanthomonas arboricola pv. pruni. BMC genomics, 22(1), 360). However, there are no reports of using PEP polypeptides, such as PEP1, as a means of delivering a cargo domain into a plant, and no reports of applying PEP polypeptides from one plant to a plant of a different species.

[0182] In some embodiments, the PEP polypeptide is a PEP1 polypeptide. In some embodiments, the PEP polypeptide is an Arabidopsis thaliana PEP1 polypeptide. In some embodiments, the PEP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In some embodiments, the PEP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to 8 (e.g., up to 1, 2, 3, 4, 5, 6, 7, or 8) amino acid substitutions. In certain embodiments, the PEP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 2.PATENT APPLICATION DOCKET NO. P14953WOOOELF '18 Polypeptides

[0183] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both is an ELF 18 polypeptide. ELF 18 is referred to as a pathogen-associated molecular pattern (PAMP), and, more specifically, a microbe-associated molecular pattern (MAMP), that elicits a pathogen defense response in plants. ELF18 is an 18-amino acid peptide derived from bacterial elongation factor Tu (EF-Tu) and was first shown to act as a PAMP in Arabidopsis in Kunze et al.2004 (The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. The Plant Cell, 16(12), 3496-3507). The receptor that mediates ELF 18 elicitation of defense pathways has been shown to be specific to Brassicaceae (Boiler and Felix 20009. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by patternrecognition receptors. Annual review of plant biology, 60, 379-406; Lacombe et al. 2010. Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance. Nature biotechnology, 28(4), 365-369). Several studies have applied ELF18 to rooting media or infiltrated it into leaves to demonstrate the elicitation of defense responses in plants in the family Brassicaceae (e.g., Dhar et al. 2021. The danger-associated peptide PEP1 directs cellular reprogramming in the Arabidopsis root vascular system. Molecules and cells, 44(11), 830-842; Wan et al. 2019. Comparing Arabidopsis receptor kinase and receptor protein-mediated immune signaling reveals BIK1 -dependent differences. New Phytologist, 221(4), 2080-2095). However, there are no reports of ELF 18 being applied as a sprayable formulation or being used to deliver a cargo protein into a cell. In addition, besides a study in which an ELF 18 receptor from Brassicaceae was transgenically transferred to a non-Brassicaceae plant (Lacombe et al 2010), there are no reports of ELF 18 being applied to non-Brassicaceae plants.

[0184] In some embodiments, the ELF 18 polypeptide comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 60%, 70%. 80%. 90%, or 95% sequence identity thereto. In some embodiments, the ELF18 polypeptide comprises the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 8 (e.g., up to 1, 2, 3, 4, 5, 6, 7, or 8) amino acid substitutions. In certain embodiments, the ELF 18 polypeptide comprises the amino acid sequence of SEQ ID NO: 3.PATENT APPLICATION DOCKET NO. P14953WOOOpLAA Fusion Proteins

[0185] In some embodiments, the polypeptide configured to enter plant cells, move within plants, or both is p(LysAibAla) (pLAA). pLAA is a synthetic cell-penetrating peptide made up of a polymerized tripeptide consisting of, from N-terminus to C-terminus, lysine (Lys)-alpha-aminoisobutyric acid (Aib)-alanine (Ala). pLAA has been synthesized with degrees of polymerization between 2 and 5 (Terada, et al. 2020. Artificial cell-penetrating peptide containing periodic a-aminoisobutyric acid with long-term internalization efficiency in human and plant cells. ACS Biomaterials Science & Engineering, 6(6), 3287-3298); however, the pLAA used herein (SEQ ID NO: 5) has a degree of polymerization of 5. pLAA has been used primarily for the delivery of nucleic acids to cells in cell culture or into leaves using syringe infiltration (e.g, Terada, et al.) and vacuum infiltration (e.g., Fujita et al. 2021. All-Peptide-based polyion complex vesicles: facile preparation and encapsulation of the protein in active form. ACS Polymers Au, 1(1), 30-38). There have been reports of spray application of pLAA to leaves to deliver nucleic acids to intact plants (Thagun et al. 2022. Non-transgenic gene modulation via spray delivery of nucleic acid / peptide complexes into plant nuclei and chloroplasts. ACS nano, 16(3), 3506-3521), and the delivery of cargo proteins complexed to pLAA via ionic interactions and non-peptide chemical linkages has been contemplated (US11505577B2). However, there are no reports of fusion proteins with pLAA covalently attached to other proteins.

[0186] In some embodiments, provided herein is a protein comprising a cargo polypeptide and pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA, wherein the cargo domain and the pLAA are covalently attached. In some embodiments, the cargo domain and the pLAA are covalently attached via a peptide bond. In certain embodiments, the protein comprises a peptide linker between the cargo domain and the pLAA. The peptide linker may be any suitable linker known in the art or described herein. In other embodiments, the cargo domain and the pLAA are covalently attached via a triazole ring (e.g. , via click chemistry).

[0187] In some embodiments, provided herein is a protein comprising protein comprising a polypeptide configured to enter the leaves of the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b), the polypeptide comprising: CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1), andPATENT APPLICATION DOCKET NO. P14953WOOOpLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA, wherein the CEP1 and the pLAA are fused via a peptide bond. In some embodiments, the protein comprises a peptide linker between the CEP1 and the pLAA. The peptide linker may be any suitable linker known in the art or described herein. In certain embodiments, the peptide linker comprises between 1 and 10 glycine and serine residues. In certain embodiments, the peptide linker comprises or consists of G4S (SEQ ID NO: 10). In some embodiments, the protein further comprises a cargo domain. In certain embodiments, the cargo domain is fused to the polypeptide via a peptide bond. In certain embodiments, the protein comprises a further peptide linker between the polypeptide and the cargo domain.

[0188] In some embodiments of the foregoing, the protein comprises, from N-terminus to C-terminus: CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1): and pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA. In certain embodiments, the protein comprises a peptide linker between the CEP1 and the pLAA. The peptide linker may be any suitable linker known in the art or described herein. In certain embodiments, the peptide linker comprises between 1 and 10 glycine and serine residues. In certain embodiments, the peptide linker comprises or consists of G4S (SEQ ID NO: 10). In some embodiments, the protein comprises, from N-terminus to C-terminus: CEP1 (SEQ ID NO: 1) or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1); a peptide linker (e.g., SEQ ID NO: 10); and pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%. 90%, or 95% sequence identity’ to pLAA. In some embodiments, the protein comprises, from N-terminus to C-terminus, CEP1 (SEQ ID NO: 1); G4S (SEQ ID NO: 10); and pLAA (SEQ ID NO: 7). In some embodiments, the protein comprises CEPl-G4S-p(LysAibAla), also referred to as CpLAA and Fl (SEQ ID NO: 8), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In certain embodiments, the protein comprises CEPl-G4S-p(LysAibAla), also referred to as CpLAA and F 1 (SEQ ID NO: 8). In some embodiments, the protein further comprises a cargo domain. In certain embodiments, the cargo domain is fused to the polypeptide via a peptide bond. In certain embodiments, the protein comprises a further peptide linker between the polypeptide and the cargo domain.

[0189] In some embodiments of the foregoing, the protein comprises, from N-terminus to C-terminus: pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95%PATENT APPLICATION DOCKET NO. PI4953WOOOsequence identity to pLAA; and CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1). In certain embodiments, the protein comprises a peptide linker between the pLAA and the CEP1. The peptide linker may be any suitable linker known in the art or described herein. In certain embodiments, the peptide linker comprises between 1 and 10 glycine and serine residues. In certain embodiments, the peptide linker comprises or consists of G4S (SEQ ID NO: 10). In some embodiments, the protein comprises, from N-terminus to C-terminus: pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA; a peptide linker (e.g., SEQ ID NO: 10); and CEP1 (SEQ ID NO: 1). or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1). In certain embodiments, the protein comprises, from N-terminus to C-terminus: pLAA (SEQ ID NO: 7); G4S (SEQ ID NO: 10); and CEP1 (SEQ ID NO: 1). In some embodiments, the protein comprises p(LysAibAla)-G4S-CEPl, also referred to as pLAAC and F2 (SEQ ID NO: 9). or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto. In certain embodiments, the protein comprises p(LysAibAla)-G4S-CEPl, also referred to as pLAAC and F2 (SEQ ID NO: 9). In some embodiments, the protein further comprises a cargo domain. In certain embodiments, the cargo domain is covalently attached to the polypeptide, for example, via a peptide bond. In certain embodiments, the protein comprises a further peptide linker between the polypeptide and the cargo domain.Cargo Domains

[0190] In some embodiments, the proteins descnbed herein comprise a cargo domain. Cargo domains include, for example, functional proteins, nucleic acids, and other molecules or elements that are essential for plant growth and / or enhance a plant trait. In some embodiments, the cargo domain is a protein domain. In some embodiments, the cargo domain is a nucleic acid. In certain embodiments, the cargo domain is DNA (e.g., plasmid DNA). In certain embodiments, the cargo is RNA (e g, an siRNA). In some embodiments, the cargo domain is a small molecule, such as a pesticide. In some embodiments, the cargo domain is a plant hormone, such as an auxin, a cytokinin, a gibberellic acid, abscisic acid, a brassinosteroid, jasmonic acid, salicylic acid, florigen, or a strigolactone.

[0191] In some embodiments, the cargo domain and the polypeptide form a complex through non-covalent interactions. Non-covalent interactions that can cause two polypeptides (e.g., a cargoPATENT APPLICATION DOCKET NO. P14953WOOOdomain and a polypeptide configured to enter plant cells and / or move within the plant) to form a complex include, for example, ionic interactions, hydrophobic interactions, hydrogen bonds, and van der Waals forces. For example, in some embodiments, the cargo protein and the polypeptide form a complex through ionic interactions. In certain embodiments, the complex is formed through positively charged amino acids of the polypeptide (e.g, lysine, histidine, and arginine residues) interacting with negatively charged amino acids of the cargo domain (e.g, as described in US11505577B2).

[0192] In some embodiments, the cargo domain and the polypeptide configured to enter plant cells, move within the plant, or both, are fused via a peptide bond. In other words, in some embodiments, the protein is a fusion protein comprising the cargo domain and the polypeptide. Accordingly, in some embodiments, the protein comprises, from N-terminus to C-terminus, the cargo domain and the polypeptide configured to enter plant cells, move within the plant, or both. In other embodiments, the protein comprises, from N-terminus to C-terminus, the polypeptide configured to enter plant cells, move within the plant, or both, and the cargo domain. In some embodiments, the protein comprises a peptide linker between the cargo domain and the polypeptide. Accordingly, in some embodiments, the protein comprises, from N-terminus to C-terminus: the cargo domain; a peptide linker; and the polypeptide configured to enter plant cells, move within the plant, or both. In other embodiments, the protein comprises, from N-terminus to C-terminus: the polypeptide configured to enter plant cells, move within the plant, or both; a peptide linker; and the cargo domain.

[0193] Any suitable peptide linker known in the art may be used, including, for example: glycine polymers (G)n, where n is an integer of at least 1 (e.g, at least one, at least 2, at least 3, at least 4, at least 5. at least 6, at least 7, at least 8, at least 9, at least 10, etc.); glycine-serine polymers (GS)n, also referred to as GS linkers, where n is an integer of at least 1 (e.g., at least one, at least 2. at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc ); glycine-alanine polymers; alanine-serine polymers; and the like. Exemplary GS linkers include, for example, GGGGS (SEQ ID NO: 10), GGGSG (SEQ ID NO: 36), GGSGG (SEQ ID NO: 37), GSGGG (SEQ ID NO: 38), GSGSG (SEQ ID NO: 39), GSSSG (SEQ ID NO: 40), SGGS (SEQ ID NO: 41), GGSG (SEQ ID NO: 42). Peptide linker sequences may be of any length, such as from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., 20 amino acid glycine polymers or glycine-serine polymers), about 1 amino acid to about 15 amino acids, about 3 amino acids toPATENT APPLICATION DOCKET NO. P14953WOOOabout 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0194] In some embodiments, the cargo domain comprises a recombinant or synthetic polypeptide. Recombinant polypeptides include, for example, a first amino acid sequence (e.g., a cargo domain) that is covalently attached (e.g., via a peptide bond) to a second amino acid sequence (e.g., a polypeptide configured to enter plant cells, move within a plant, or both), wherein the first and second amino acid sequences do not natively occur in the same polypeptide. In certain embodiments, the cargo domain comprises a recombinant polypeptide comprising a first amino acid sequence, and the polypeptide configured to enter plant cells, move within a plant, or both comprises a second amino acid sequence, wherein the first and second amino acid sequences do not natively occur in the same polypeptide. In certain embodiments, the cargo domain comprises a first amino acid sequence and a second amino acid sequence, wherein the first and second amino acid sequences do not natively occur in the same polypeptide. Synthetic polypeptides include, for example, polypeptides that do not occur in nature; polypeptides that are comprise non-natural amino acid sequences; polypeptides selected from a random polypeptide library; rationally designed non-natural amino acid sequences; polypeptides generated by artificial intelligence; and the like.

[0195] In some embodiments, the cargo domain comprises an amino acid sequence that is heterologous relative to the polypeptide configured to enter plant cells, move within a plant, or both. In some embodiments, the cargo domain comprises an amino acid sequence derived from a different source (e.g., is from a different source organism, or is a synthetic or recombinant polypeptide) than the sequence of the polypeptide configured to enter plant cells, move within the plant, or both. For example, in some embodiments, the cargo domain is derived from a different family, genus, and / or species than the polypeptide configured to enter plant cells, move within the plant, or both. In certain embodiments, the polypeptide comprises an amino acid sequence derived from a prokaryote (e.g.. a bacterium, the source organism for ELF18), and the cargo domain comprises amino acid sequence derived from a eukaryote. In certain embodiments, the polypeptide comprises an amino acid sequence derived from a bacterium, and the cargo domain comprises amino acid sequence derived from a different bacterium. In certain embodiments, the polypeptidePATENT APPLICATION DOCKET NO. P14953WOOOcomprises an amino acid sequence derived from a bacterium, and the cargo domain comprises or consists of a synthetic, non-natural, or recombinant amino acid sequence. In other embodiments, the polypeptide comprises an amino acid sequence derived from a plant, and the cargo domain comprises an amino acid sequence from an organism (e.g., a plant, bacterium, or other organism) of a different family, genus, and / or species. In certain embodiments, the polypeptide comprises an amino acid sequence derived from a plant, and the cargo domain comprises or consists of a synthetic, non-natural, or recombinant amino acid sequence.

[0196] In some embodiments, the cargo domain comprises a plant signaling polypeptide. Proteins comprising polypeptide configured to enter plant cells, move within a plant, or both and a plant signaling polypeptide cargo domain may be useful for, for example, enhancing the mobility' and / or cell penetrating ability of the protein in a plant, activating or repressing a signaling pathway within a plant, and the like. The plant signaling polypeptide may be any plant signaling polypeptide known in the art or described herein. For example, in some embodiments, the cargo domain comprises a CEP polypeptide. In certain embodiments, the cargo domain comprises CEP1 (SEQ ID NO: 1), or an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity thereto.

[0197] In some embodiments, the cargo domain comprises an antigen-binding polypeptide. Proteins comprising a polypeptide configured to enter plant cells, move within a plant, or both an antigen-binding cargo domain may be useful for, for example, for binding to, and activating or inhibiting, a target protein in a plant. The target protein may be an enzyme, a signaling polypeptide, or any other protein in the plant. Antigen-binding polypeptides include, for example, antibodies and antibody fragments. Antibody fragments may comprise one or more antibody heavy chain variable (VH) domains, one or more antibody light chain variable (VL) domains, or both. The antibody fragment may be any format of antibody fragment known in the art, for example, a Fab, aFab’, aF(ab)2, a single-domain antibody (e.g., aVHH), an Fc fragment, an Fv, an scFv, adiabody, a triabody, and the like. In certain embodiments, the cargo domain comprises a single-domain antibody. In certain embodiments, the cargo domain comprises a VHH. Antibodies may be full-length antibodies, comprising VH. VL, and Fc domains. Antibodies may be monoclonal or polyclonal and may be produced by suitable methods known to those of skill in the art.PATENT APPLICATION DOCKET NO. PI4953WOOO

[0198] In some embodiments, the cargo domain comprises a site-directed nuclease. Proteins comprising polypeptide configured to enter plant cells, move within a plant, or both and a site-directed nuclease may be useful for, for example, genetically modifying a plant, plant, part, or plant cell through gene editing and / or transgenesis. Site-directed nucleases include, for example, RNA-guided nucleases (e.g., Cas nucleases), TALENS, zinc finger nucleases, meganucleases, argonaute nucleases, and the like.

[0199] In some embodiments, the cargo domain comprises a transcription factor. Proteins comprising polypeptide configured to enter plant cells, move within a plant, or both and a transcription factor may be useful for, for example, upregulating or downregulating the expression of a target gene or pathway within a plant.

[0200] In some embodiments, the cargo domain comprises a pesticidal protein. Pesticidal proteins include, for example, herbicidal proteins, fungicidal proteins, insecticidal proteins, nematocidal proteins, and bacteriocidal proteins. Proteins comprising polypeptide configured to enter plant cells, move within a plant, or both and a pesticidal protein may be useful for, for example, delivering to a plant a protein that can move within the plant and allow protection against pathogens and / or herbivores such as insects. Pesticidal proteins include, for example, proteins from Bacillus thuringensis (e.g., Cry, Cyt, Vip, and Sip proteins), Mtx proteins, lectins, protease inhibitors, and the like.

[0201] In some embodiments, the cargo domain comprises an herbicidal protein. Proteins comprising a polypeptide configured to enter plant cells, move within a plant, or both and an herbicidal protein may be useful for, for example, protein-based control of w eeds in an agricultural setting.

[0202] In some embodiments, the cargo domain is a protein that enhances a plant trait. In some embodiments, the plant trait is yield. In certain embodiments, the plant trait is yield potential. In some embodiments, the plant trait is resistance to an abiotic stressor. Abiotic stressors include, for example, extreme temperatures, drought, flooding, high salinity, and the like. In some embodiments, the plant trait is drought tolerance. In some embodiments, the plant trait is heat tolerance. In some embodiments, the plant trait is frost resistance. In some embodiments, the plant trait is nutrient use efficiency. In certain embodiments, the plant trait is nitrogen use efficiency. In some embodiments, the plant trait is a quality trait. Quality traits include, for example, flavor,PATENT APPLICATION DOCKET NO. PI4953WOOOtexture, nutritional content, shelflife, color, size, and the like, and can be traits of any part of the plant, for example, a tuber, a fruit, a flower, a stem, and the like.

[0203] In some embodiments, the cargo domain comprises or consists of an element or molecule that is essential for plant growth, for example, a nutrient. Nutrients include both macronutrients (nitrogen, phosphorus, potassium, sulfur, and magnesium) and micronutrients (e.g., copper, zinc, molybdenum, boron, iron, manganese, chlorine, and nickel). Nutrients are necessary7for plant metabolic processes, for example, biosynthesis of nucleic acids and proteins (e.g., nitrogen and sulfur); biosynthesis of porphyrins such as chlorophyll, heme, and leghemoglobin (e.g, iron, magnesium, manganese); and biosynthesis of secondary7metabolites such as flavonoids (e.g, zinc, copper, magnesium, and iron), phenolic compounds (e.g, manganese and iron), terpenoids (e.g, iron), and alkaloids (e.g. copper and zinc). In certain embodiments, the cargo domain comprises a molecule that is rich in a macronutrient or micronutrient. In some embodiments, the cargo domain is a nanoparticle comprising a nutrient or anti -microbial element, for example, copper, iron, magnesium, manganese, silver, titanium, or zinc. In certain embodiments, the cargo domain comprises or an amino acid sequence designed to alleviate nutritional deficiencies in the plant, or a grain or seed derived therefrom.Plants and Plant Parts

[0204] In some embodiments, provided herein is a plant, plant part, or plant cell comprising one or more of the proteins described herein. The plant, plant part, or plant cell may be of any plant order, family, genus, or species described herein. In some embodiments, the plant is a monocot. In other embodiments, the plant is a dicot.

[0205] In some embodiments, the plant, plant part, or plant cell is of a different plant order, family, genus, or species than the source organism of the protein or a part thereof (e.g, the cargo domain and / or the polypeptide configured to entered plant cells, move within the plant, or both). The protein or part thereof may be from any source organism described herein. In some embodiments, the plant is a monocot and the source organism is a dicot.

[0206] In some embodiments, the plant cell is a protoplast. In some embodiments, the plant part is a leaf, stem, flower, fruit, or root that has been excised from a plant. In certain embodiments, thePATENT APPLICATION DOCKET NO. PI4953WOOOplant part is an excised leaf. In some embodiments, the plant part is plant tissue in plant tissue culture. In some embodiments, the plant part is a callus.

[0207] In some embodiments, the plant comprising one or more of the proteins described herein is an intact plant. In some embodiments, the intact plant is present in an agricultural field. In some embodiments, the intact plant is a food or industrial crop, such as, for example, maize, rye, alfalfa, apple, artichoke, asparagus, avocado, banana, barley, basil, beans (e.g., soybeans, common beans, and the like), beets, berries, blackberry, blueberry, broccoli, brussels sprouts, cabbage, cacao, carrot, cashew, cassava, cauliflower, celery, chard, chickpea, chives, citrus (e.g., orange, lemon, lime, grapefruit, citron, and the like), clover, coffee, collards, cotton, cranberry, cucumber, currant, eggplant, fig, flax, garlic, ginger, grape, guava, hazelnut, hemp, hop, kale, kiwi, kohlrabi, leek, lentil, lettuce, maple, melon, millet, miscanthus, mustard, oat, oil palms, okra, olive, onion, papaya, parsnip, peanut, pea, pear, penny cress, pepper (e.g.. sweet peppers, hot peppers, and the like), peppercorn, pine, pineapple, pistachio, pomegranate, poplar, potato, quinoa, radish, rapeseed, raspberry', rice, rose, safflower seed, scallion, sesame, shallot, sorghum, soybean, spinach, squash, stone fruit (e.g., cherry, peach, plum, apricot, nectarine, almond, and the like), strawberry', sunflower, sugarcane, sweet potato, switchgrass, tea. teff, tobacco, tomatillo, tomato, turf grass, turnip, walnut, wheat, yams, or yucca. In certain embodiments, the intact plant is a rye plant. In certain embodiments, the intact plant is a maize plant. In some embodiments, the intact plant is a weed, for example, any weed species described herein. In certain embodiments, the intact plant is Palmer amaranth.ENUMERATED EMBODIMENTS

[0208] Various exemplary embodiments of the proteins, molecules, plants, plant parts, genomes, chromosomes, methods, biological samples, and other compositions described herein are set forth in the following set of numbered embodiments.

[0209] 1. A composition comprising a protein configured to penetrate the cuticle of a plant, enter plant cells, and move systemically within the plant when applied to the surface of an intact plant.

[0210] 2. A method of delivering a protein into a plant, comprising:PATENT APPLICATION DOCKET NO. P14953WOOO

[0211] (a) applying a protein-containing composition to an intact plant, wherein the protein is configured to penetrate the cuticle of the plant, enter plant cells, and move systemically within the plant.

[0212] 3. A method of identifying proteins capable of systemic movement within a plant, comprising:

[0213] (a) testing candidate proteins for the ability to penetrate the cuticle of an intact plant,

[0214] (b) testing the candidate proteins for entry into plant cells, and

[0215] (c) evaluating the systemic movement of the proteins within the plant.

[0216] 4. The composition of embodiment 1, wherein the protein is a recombinant protein.

[0217] 5. The composition of embodiment 1. further comprising a surfactant to enhance cuticle penetration.

[0218] 6. The composition of embodiment 1, wherein the protein is fused to a targeting peptide to enhance systemic movement within the plant.

[0219] 7. The composition of embodiment 1, wherein the protein is a plant-derived protein.

[0220] 8. The composition of embodiment 1. wherein the protein comprises a cargo domain for delivering molecules into plant cells.

[0221] 9. The composition of embodiment 1, wherein the protein is selected from the group consisting of transport proteins, signaling proteins, and enzymes.

[0222] 10. The method of embodiment 2, wherein the composition is applied via foliar spray.

[0223] 11. The method of embodiment 2, wherein the composition is applied via irrigation water.

[0224] 12. The method of embodiment 2, wherein the protein is applied in combination with a nutrient or pesticide.

[0225] 13. The method of embodiment 2, wherein systemic movement of the protein is facilitated by phloem transport.PATENT APPLICATION DOCKET NO. P14953WOOO

[0226] 14. The method of embodiment 2, further comprising monitoring protein uptake using a labeled version of the protein.

[0227] 15. The method of embodiment 2. wherein the protein enhances plant resistance to insect pests, disease, or abiotic stress.

[0228] 16. The method of embodiment 3, wherein candidate proteins are screened using a high-throughput assay.

[0229] 17. The method of embodiment 3, wherein systemic movement is evaluated using fluorescence or radiolabeled tracking.

[0230] 18. The method of embodiment 3, wherein candidate proteins are derived from a protein library generated by random mutagenesis.

[0231] 19. The method of embodiment 3, wherein proteins identified are further modified to enhance cuticle penetration.

[0232] 20. The method of embodiment 3, further comprising incorporating identified proteins into an agricultural formulation.

[0233] 21. A method for delivering a protein into a plant, comprising spraying a composition containing the protein onto the plant, wherein the protein penetrates the plant cuticle and enters the plant cells.

[0234] 22. A method for delivering a protein into a plant, comprising spraying a composition containing the protein onto an intact plant, wherein the protein penetrates the plant cuticle, enters the plant cells, and moves systemically within the plant.

[0235] 23. A method for delivering a protein into a plant, comprising applying a composition containing the protein to the soil, wherein the protein is taken up by the plant roots and moves systemically within the plant.

[0236] 24. The method of embodiment 21, wherein the protein is fluorescently labeled with TAMRA.PATENT APPLICATION DOCKET NO. P14953WOOO

[0237] 25. The method of embodiment 21, wherein the protein is selected from the group consisting of AtNSl, ELF18, Pepl, Systemin, TobHypSys2, TobHypSysl, pLysAibALA (pLAA), Cep-G4S-pLAA, and pLAA-G4S-Cep.

[0238] 26. The method of embodiment 21, wherein the composition further comprises a surfactant to enhance protein penetration.

[0239] 27. The method of embodiment 21, wherein the plant is an Arabidopsis plant.

[0240] 28. The method of embodiment 21, wherein the protein is applied as a droplet to a single leaf of the plant.

[0241] 29. The method of embodiment 22, wherein the protein is fluorescently labeled with TAMRA.

[0242] 30. The method of embodiment 22. wherein the protein is selected from the group consisting of AtNSl, ELF18, Pepl, Systemin, TobHypSys2, TobHypSysl, pLysAibALA (pLAA), Cep-G4S-pLAA, and pLAA-G4S-Cep.

[0243] 31. The method of embodiment 22, wherein the composition further comprises a surfactant to enhance protein penetration.

[0244] 32. The method of embodiment 22, wherein the plant is an Arabidopsis plant.

[0245] 33. The method of embodiment 22, wherein the protein is applied as a droplet to a single leaf of the plant.

[0246] 34. The method of embodiment 23, wherein the protein is fluorescently labeled with TAMRA.

[0247] 35. The method of embodiment 23, wherein the protein is selected from the group consisting of AtNSl. ELF18, Pepl, Systemin, TobHypSys2. TobHypSysl, pLysAibALA (pLAA), Cep-G4S-pLAA, and pLAA-G4S-Cep.

[0248] 36. The method of embodiment 23, wherein the composition further comprises a surfactant to enhance protein penetration.PATENT APPLICATION DOCKET NO. P14953WOOO

[0249] 37. The method of embodiment 23, wherein the plant is an Arabidopsis plant.

[0250] 38. The method of embodiment 23, wherein the protein is applied as a droplet to a single leaf of the plant.

[0251] 39. A method for discovering proteins capable of penetrating the plant cuticle, entering plant cells, and moving systemically within the plant, comprising applying a fluorescently labeled protein to an intact plant and recording fluorescent and brightfield images at multiple times after application.

[0252] 40. The method of embodiment 39, wherein the fluorescent label is TAMRA.EXAMPLESExample 1: Experimental Peptides Application

[0253] Methods:

[0254] Experimental proteins were fluorescently labeled with TAMRA according to the manufacturer's directions and resuspended in water as a lOmg / ml stock solution.

[0255] 1 pl (lOpg) of experimental peptide was applied to the leaf of an intact Arabidopsis plant on soil, as a droplet, and its position was noted and allowed to air dry in the growth chamber.

[0256] Fluorescent and brightfield images were recorded at multiple times after application. Initial Experimental Peptides:

[0257] Arabidopsis Origin Signal Peptides: AtNSl (also referred to as CEP1 or CEP; SEQ ID NO: 1), Pepl (also referred to as PEP; SEQ ID NO:2).

[0258] Bacterial Origin Elicitor Peptide: ELF 18 (SEQ ID NO: 3).

[0259] Solanaceous Origin Signal Peptides: Systemin (also referred to as SYS or LeSl; SEQ ID NO: 4), TobHypSys2 (also referred to as THSII, THS2 or NtS2; SEQ ID NO: 6), TobHypSysl (also referred to as THSI, THS1 orNtSl; SEQ ID NO: 5).PATENT APPLICATION DOCKET NO. P14953WOOO

[0260] Plant Demonstrated CPP in culture and with injection: pLysAibALA (pLAA; SEQ ID NO:7).

[0261] Combinations: Cep-G4S-pLAA (CEP fused to N-terminus of pLAA via G4S linker, also referred to as CpLAA or Fl; SEQ ID NO: 8), pLAA-G4S-Cep (pLAA fused to N-terminus of CEP via G4S linker, also referred to as pLAAC or F2; SEQ ID NO: 9).

[0262] Results:

[0263] FIG. 1: Peptides applied to leaf discs.

[0264] FIGS. 2A and 2B: Fluorescent image showing the expansion of fluorescence from the application point on DI for many peptides, particularly evident in Solanaceous sourced and combinations.

[0265] FIGS. 3 and 4: Fluorescent images showing evidence for systemic movement with multiple peptides.Example 2: Sprayable Proteins Penetration and Movement

[0266] Methods: Proteins capable of penetrating the plant cuticle, entering plant cells, and moving systemically within the plant will be applied through spray application.

[0267] Results: The proteins will demonstrate effective penetration and movement within the plant, providing a non-invasive method for introducing functional proteins into plants for agricultural, horticultural, or biotechnological applications.

[0268] Additional Figure(s): Brightfield image showing the systemic movement of proteins within the plant.Example 3: Application of Sprayable Proteins for Enhanced Plant Growth and Disease Resistance

[0269] Objective: To demonstrate the effectiveness of sprayable proteins in penetrating the plant cuticle, entering plant cells, and moving systemically within the plant to enhance growth and disease resistance.PATENT APPLICATION DOCKET NO. PI4953WOOO

[0270] Methods

[0271] Protein Preparation: A protein known for its growth-promoting and disease-resistance properties, such as AtNSl, will be fluorescently labeled with TAMRA to allow for visualization. The protein will then be formulated into a sprayable composition with a surfactant to enhance penetration.

[0272] Plant Selection: Intact Arabidopsis plants will be chosen for the experiment due to their well-characterized genetics and ease of handling.

[0273] Application: The spray able protein composition will be applied to the leaves of the intact Arabidopsis plants using a fine mist sprayer. The application will be performed in a controlled growth chamber to ensure consistent environmental conditions.

[0274] Observation: Fluorescent and brightfield images will be recorded at multiple time points (e.g., 1 hour, 24 hours. 48 hours, and 72 hours) after application to monitor the penetration, entry, and systemic movement of the protein within the plant.

[0275] Soil Application: In a parallel experiment, the same protein composition will be applied to the soil around the roots of the Arabidopsis plants. The uptake and systemic movement of the protein will be monitored using similar imaging techniques.

[0276] Expected Results:

[0277] Leaf Application: The fluorescent images are expected to show that the TAMRA-labeled protein successfully penetrates the plant cuticle and enters the plant cells within 1 hour of application. By 24 hours, the protein is anticipated to move systemically within the plant, reaching distant leaves and stems. The brightfield images are expected to confirm that the plants exhibit enhanced growth and increased resistance to a common pathogen, Pseudomonas syringae.

[0278] Soil Application: The fluorescent images are expected to demonstrate that the protein is taken up by the plant roots and moves systemically within the plant. Similar to the leaf application, the protein is anticipated to reach distant parts of the plant within 24 hours, promoting growth and enhancing disease resistance.PATENT APPLICATION DOCKET NO. PI4953WOOOExample 4: Confirmation of Entry and Movement of Peptides in Arabidopsis thaliana

[0279] This example further demonstrates entry of the peptides described in Example 1 into leaf vasculature and leaf cells in Arabidopsis (A. thaliana) leaf discs and leaves or intact Arabidopsis plants.Example 4A: Leaf Discs

[0280] Each peptide described in Example 1 was labeled with the fluorophore TAMRA at a ratio of one TAMRA molecule per one peptide molecule. One mg of each peptide was diluted in 100 pL of water to a concentration of lOmg / mL. For leaf disc experiments, all peptides were diluted to 0.25 mg / mL in a final volume of 500 pL. corresponding to 125 pM working solution for a 2000 g / mol peptide.

[0281] Six leaf discs from 32-day old Arabidopsis Col-0 plants were harvested into 12 well plates. Discs were incubated abaxially (upside down) in the peptide working solutions for 1.5 hours. The dye was removed by pipetting and washed twice with 3 mL of water, mixing three times. Each disc was then returned to the plate and incubated in 700 pL of water for a half hour. Finally, the discs were placed adaxially (right side up) in the well and imaged at lOx magnification using a dissecting microscope (Lieca M165FC).

[0282] As shown in FIGs. 6A-6B, all peptides were visible in the vascular tissue of the leaf discs, confirming the results demonstrated in Example 1. Most of the peptides are visible in the vasculature, including CEP, SYS, THSI, ELF, and PEP. Globular fluorescence is visible in discs incubated with ELF, PEP, CpLAA, pLAA, and pLAAC also show globular fluorescence within the leaf disc, suggesting penetration of cells within the disc.Example 4B: Leaves of Intact Arabidopsis Plants

[0283] This example further demonstrates entry' of the peptides described in Example 1 into the leaf vasculature and leaf cells in intact leaves of A. thaliana plants.

[0284] Arabidopsis plants were grown in 98 cell flats for 19 days (d). For each of the TAMRA-labeled peptides from Example 1. 5 pL of 2.5 mg / mL of each peptide was applied to the adaxial (upper) an intact leaf in several droplets, increasing the coverage of the leaf with the same mass of peptide as in the experiment in Example 1. The plants were placed in a laminar flow' hood to dry and were imaged at 3hr, Id, 2d, 3d, and 6d after application of the peptides to the leaves. Images were captured on a dissecting stereoscope with an ET-DSR filter to capture TAMRA signal. Z-PATENT APPLICATION DOCKET NO. P14953WOOOstacks were used with the lowest focal plane at the base of the petiole and the highest focal plane at the highest point of the load leaf, LASX softw are determined the best number of focal slices. Peptides were evaluated based on the strength of the TAMRA signal originating from the vasculature and the distance / spread the peptides exhibited in the leaf. For each peptide, a bright field and three ET-DSR exposures were taken: moderate exposure low gain, low exposure moderate gain, and high exposure and gain. All figures were made with the high exposure and gain. In order to capture the vascular localization of the peptides, long exposures (1 second) and high gain settings were used. Exposure times on later timepoints were increased to show spread of TAMRA-labeled peptide within the plant and in close range by the application leaf.

[0285] As shown in FIGS. 7A-7C. fluorescence in vasculature and movement in the apoplast away from the application site was observed after treatment with several of the peptides, including SYS, THSII, ELF, PLAA, CPLAA, PLAAC, and THSI. In at least pLAAC, fluorescence is also observed in the trichomes (FIG. 7C, white arrows), which are living cellular structures, indicating that the TAMRA-labeled peptide entered into plant cells.

[0286] A third experiment with intact Arabidopsis plants was performed as described above, with modification. First, the peptides were applied using a single droplet of 1 [1L of peptide solution at 0.25 mg / mL, a lower concentration than the previous intact leaf experiments. In addition, plants were grown in 32-cell flats, resulting in plants that were larger than the plants than those grown in 98-cell flats for the previous experiments. Plants were imaged at 1, 3, and 5d.

[0287] As can be observed in FIGs. 8A-8C, the increased height of the larger plants led to less clear imaging of the leaves. Still, plants treated with the CEP, SYS, THSII, ELF, PEP, and PLAAC peptides exhibited fluorescence in the vasculature of the leaves distal of the application site, with at least THSII and PLAAC exhibiting diffuse fluorescence between the vasculature, suggesting entry into leaf cells (FIGS. 8A-8C), suggesting that these peptides are able to enter the leaf and move within the plant even when applied at lower concentrations.Example 4C: Treatment of Arabidopsis with Different Peptide Concentrations

[0288] An experiment w as conducted in w hich the leaves of intact Arabidopsis plants were treated with various concentrations of the peptides. One 38 cell flat of Promix FPX was directly sown with Arabidopsis var. Col-0 and grown in a Conviron set to 24°C. At emergence each cell was thinned to contain one plant. Plants were treated with the peptides at 16 days after sowing (DAS) andPATENT APPLICATION DOCKET NO. PI4953WOOOimaged 19 DAS. The ELF and pLAA peptides were selected for testing the effect of reduced concentration on uptake and movement. A water control, but no free TAMRA control was made. Four concentrations were selected, 1.00, 0.50, 0.25, and 0.10 mg / mL. These were prepared in serial dilution. At the base of the V3 petiole 1 pL of a Tween-20 solution (1 drop in 1.5 mL) was applied prior to the addition of 1 pL of diluted peptide. The peptide solution was briefly allowed to dry in the air before the flat was transferred to a Conviron for three days. The plant treated with water and tween was marked with a marker below the treatment location.

[0289] For imaging, treated leaves were excised from the rosette and the base taped to a microscope slide. After flattening the leaf, a second slide was placed atop the leaf. This leaf sandwich was placed on top of a third slide to raise the focal plane of interest above the bottom of the dissecting scopes working optical range. Some leaves were curled or formed a dome shape. In this case the leaves were cut with a razor blade to allow- them to lay flat. Images w ere collected in bright field and using an ET-DSR filter at a 0.7x magnification using the same setting for all images. The ET-DSR settings were the medium gain / exposure used previously. The fluorescent and brightfield images are shown in FIGS. 9A-9D.

[0290] In plants treated with ELF, branching TAMRA signal to either or both marginal sides of the leaf was observed (FIG. 9C). However, litle difference was observed between the application concentrations of ELF, and the signal intensity7did not visibly correlate with peptide concentration. In contrast, pLAA showed a wide range of signal intensity7, with strong uptake and movement across the entire leaf body and clear correlation between signal intensity and concentration (FIG.9D).Conclusion

[0291] Taken together, these results exemplify peptides that are able to enter and move within the vasculature of Arabidopsis and in some instances enter plant cells. Leaf disc experiments replicated the results in Example 1 by showing that all peptides tested were capable of moving within the vasculature of Arabidopsis leaves, and suggesting that ELF, PEP, CpLAA, pLAA, and pLAAC are able to enter leaf cells (FIGS. 1 and 6A-6B). Treatment of intact Arabidopsis plants showed that several peptides were able to enter the plant epidermis and move within the vasculature of leaves (FIGS. 7A-8C). THSII / NtS2, SYS, ELF, and pLAAC / F2 consistently exhibited mobility in Arabidopsis leaves across these experiments and Example 1 (FIGS. 2-4 and 7-8), with THSII exhibiting particularly strong fluorescence and movement across all experiments (FIGS. 2A, 3, 7A,PATENT APPLICATION DOCKET NO. PI4953WOOOand 8A). pLAA / FCompl also showed strong movement and fluorescence across all but one experiment (FIGS. 2A, 3, 7B, 8B, and 9D).

[0292] These results are surprising, at least because the majority of the peptides, including those that showed the most consistent movement, are not native to Arabidopsis. For example, THSII / NtS2 is a plant signaling peptide derived from tobacco (Pearce et al. 2001. Production of multiple plant hormones from a single polyprotein precursor. Nature, 411(6839), 817-820). SYS is a plant signaling peptide derived from tomato (Pearce et al. 1991. A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science. 253(5022): 895-7), and ELF is an elicitor peptide derived from bacterial elongation factor TU (Kunze et al. 20004. The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. The Plant Cell, 16(12), 3496-3507). Prior to these experiments, a person of ordinary skill in the art would not have expected that these peptides are able to enter Arabidopsis leaves and cells and move within Arabidopsis plants.

[0293] In addition, it is notable that both the CpLAA / Fl and pLAAC / F2 fusion proteins, which both comprise of the cell-penetrating peptide pLAA fused to CEP (AtNSl) from Arabidopsis, showed more consistent and stronger movement than CEP from Arabidopsis alone (FIGS. 2A, 3, 7A-7B, and 8A-8B). These results suggest that the cell-penetrating peptide pLAA may be fused to a less-mobile cargo protein (e.g, CEP) to allow penetration and movement of the cargo protein within the plant. In addition, pLAAC was more consistently mobile than pLAA, suggesting some synergy between pLAA and CEP in the fusion protein to allow for more consistent movement within the plant.Example 5: Entity and Movement of Peptides Leaves of Intact Palmer Amaranth Plants Example 5A: Initial Experiment

[0294] This example demonstrates entry of peptides into the leaf vasculature and leaf cells in intact leaves of Palmer amaranth (Amaranthus palmeri) plants.

[0295] An uptake / movement assay was conducted using all 15 (mono and oligo) peptides and free TAMRA using plants 11 DAS. Two peptides were applied to each plant on the first fully expanded leaf pair (one peptide per leaf) to observe penetration and movement of each peptide within a single leaf. Plants were initially grown in 2.5” deep pots which resulted in plants which were too tall to fit in the dissecting scope. They were repotted into 3.5” pots, attempting to minimize rootPATENT APPLICATION DOCKET NO. P14953WOOOdisturbance. No reduction in growth was apparent as a result. Each plant was imaged 12hr and 3d after application; a subset of the Amaranth were imaged 5d after application.

[0296] One pL of ~1 pg / pL of each peptide was spotted onto each leaf and was not disturbed until all peptides were visibly dry. Leaves were tagged with a small piece of blue tape to identify which leaf had a peptide applied to it. Plants were imaged using the dissecting microscope used previously (Lieca M165FC). The results are shown in FIG. 10.

[0297] SYS (FIG. 10C) exhibited a moderate signal which flowed from the application point to the distal leaf tip, indicating movement within the plant away from the application site. The fluorescence was observed in a ‘V’ shape, meaning that the fluorescence cannot be explained by the peptide solution ‘running’ dow n the leaf surface. Interestingly, proportionately little signal w as observed in the veins with most of the signal originating from globular bodies, suggesting intracellular penetration of the peptides. THSII (FIG. 10F) behaved similarly to SYS except that the signal was observed on only on the one side of the leaf. pLAAC (FIG. 1 OH) show ed extremely high signal in an asymmetric V, opening towards the distal leaf surface.Example 5B: Experiment with Tween Pre-Treatment

[0298] A second experiment was conducted in which the Amaranth leaves were pre-treated with Tween. Amaranth was sown in Promix and grown at 27°C watering as needed. The plants were thinned at approximately 8 DAS, two 38 cell flats of plants were sown to yield 17 suitable plants. VI leaves of Amaranth plants were treated at 11 DAS. preferring fully expanded leaves. Leaves were prepared by applying 1 pL of a solution composed of 1 drop of tween-20 per 1.5 pL water. One pL of each peptide (1 mg / mL for all) was then spotted onto the droplet of tween. The tw een and peptides were applied ~7mm from the petiole junction. Three days after application the leaves were imaged. To image the peptides, the treated leaf was excised from the stem and pressed between two slides. For some samples the petiole was removed from the leaf to allow the leaf to lay flat on the slide. When needed, additional images of the top and bottom of the leaf were taken. The results are shown in FIGS. 11 A-l IK.

[0299] As before, treatment with several of the peptides tended to result in a one- or two-sided V-shaped pattern of fluorescence moving distally from the application site. SYS treatment resulted in clear globular fluorescence between the vasculature on both the top and bottom half of the leaf treated with SYS (FIG. 1 IF). Leaves treated with THSII (FIG. 11G) and pLAAC (FIG. 11 J) bothPATENT APPLICATION DOCKET NO. PI4953WOOOhad strong fluorescence in leaf vasculature. Treatment with THSII also resulted in globular patterns of fluorescence between leaf vasculature, suggesting efficient intracellular penetration by THSII (FIG. 11G). pLAA resulted in strong fluorescence distal from the application site, both in and between the leaf vasculature, suggesting efficient uptake, movement, and cellular penetration by pLAA (FIG. UK).Conclusion

[0300] Taken together, these results demonstrate penetration and movement within Palmer amaranth leaves by several peptides. In both experiments, SYS (FIG. 10C and 1 IF) and THSII (FIG. 10F and 11G) both exhibited fluorescence moving distally from the application site, suggesting that these peptides both penetrate and move in Palmer amaranth leaves. In addition, pLAAC showed strong fluorescence in the vasculature in both experiments (FIGS. 10H and 11 J), and very strong diffuse fluorescence across the leaf in FIG. 10H. pLAA showed strong fluorescence in the vasculature as well as diffuse fluorescence across the leaf in FIG. 11 K, and also some fluorescence in the vasculature in FIG. 101. PEP (FIGs. 10G and 11H) also showed some fluorescence in the vasculature outside the application site, suggesting mobility' within Amaranth leaves.

[0301] Similar to Example 5, these results are surprising, given that none of the peptides tested are from Palmer amaranth, or even from the Amaranthaceae family. The fact that both SYS (tomato) and THSII (tobacco) demonstrated consistent movement in Palmer amaranth as well as Arabidopsis (Example 4) suggests that these proteins may be applicable across a variety' of plant species. In addition, the strong fluorescence and movement of pLAAC in Palmer amaranth, compared to CEP alone (FIGS. 10A and 1 ID), further confirms that a less-mobile cargo protein (CEP) may be carried into a plant by another more-mobile protein (pLAA) and suggests a synergy between CEP and pLAA.Example 6: Penetration and Movement in Leaves of Intact Rye Plants

[0302] This example demonstrates entry of peptides into the leaf vasculature and leaf cells in intact leaves of winter rye (Secale cereale) plants.Example 6A: Initial Experiment

[0303] An initial uptake / movement assay was conducted in rye using several TAMRA-labeled peptides and free TAMRA using plants 11 DAS. Rye leaves form a helical structure, so they werePATENT APPLICATION DOCKET NO. P14953WOOOweighed down with pipete tips before peptide application to avoid movement of the droplet on the leaf surface after application (FIG. 12A). One pL of ~1 ug / pL of each peptide was spoted onto each leaf and was not disturbed until all peptides were visibly dry. Leaves were tagged with a small piece of blue tape to identify which leaf had a peptide applied to it. Plants were imaged using the dissecting microscope used previously (Lieca M165FC). Each plant was imaged 12hr and 3d after application. During imaging, glass slides were used. Untreated leaves were used as negative controls to show7background fluorescence (FIG. 7L). The results are shown in FIGS. 12A-12L.

[0304] CEP showed strong fluorescence across the length and span of the leaf distal of the application site (FIG. 12B, Day 1, top images). Within 12 hr (Day 1) strong fluorescence w as also visible in the leaf tip (FIG. 12B, Day 1, botom image). SYS produced a diffuse and homogenous signal distal to the application site (FIG. 12C). Plants treated with THSI (FIG. 12E), THSII (FIG.12F), PEP (FIG. 12G), and pLAA (FIG. 12G) all show strong fluorescence starting at the application site and moving distally.Example 6B: Experiment with Tween Pretreatment

[0305] A second experiment was conducted in which the rye leaves were pre-treated with Tween, in order to reduce the hydrophobicity7of the leaf cuticle and allow7cleaner application of the droplet. Rye was sown in Promix and grown at 27°C, watering as needed. Cotyledons were treated at 6 DAS, preferring fully expanded leaves. Leaves were prepared by applying 1 pL of a solution composed of 1 drop of tween-20 per 1.5 pL w ater. One pL of each peptide (1 mg / mL for all) was then spoted onto the droplet of tw een. Tw een and peptides w ere applied ~l-2” from the leaf tip. Three days after application the leaves were imaged. To image the peptides, the treated leaf was excised from the stem and pressed between two slides. Images were taken of the adaxial leaf surface for all peptides, and an abaxial image was also taken for SYS. The results are shown in FIG. 13A-13L.

[0306] Rye leaves treated with SYS had fluorescence distal from the application site on both the adaxial (FIG. 13D) and abaxial (FIG. 13E) surface of the leaves. Leaves treated with THSII showed strong fluorescence far from the application site, especially on the leaf margins, including at the margins very near the leaf tip (FIG. 13G). Treatment with pLAA led to strong fluorescence distal of the application site, visible in several distal sections of the leaf (FIG. 13L).Example 6C: Treatment with Different Peptide ConcentrationsPATENT APPLICATION DOCKET NO. PI4953WOOO

[0307] An experiment was conducted in which the leaves of intact rye plants were treated with various concentrations of the peptides.

[0308] Two rye seeds were directly sown in 38 cell flats in Promix FPX and thinned at emergence. At 9 DAS the V 1 leaves were sufficiently expanded for experimentation. ELF and pLAA peptides were selected for testing the effect of reduced concentration on uptake and movement. A water control, but no free TAMRA control was made. Four concentrations were selected, 1.00, 0.50, 0.25, and 0.10 mg / mL. These were prepared in serial dilution. Approximately 2 inches from the leaf tip 1 pL of a Tw een-20 solution (1 drop in 1.5 mL) was applied prior to the addition of 1 pL of peptide.

[0309] To flatten the leaves for imaging each treated leaf was cut ~lcm below the peptide residue and the base taped to a microscope slide. After flattening the leaf, a second slide was placed atop the leaf. This leaf sandwich was placed on top of a third slide to raise the focal plane of interest above the bottom of the dissecting scopes w orking optical range. Images were collected in bright field and using an ET-DSR filter at a lx magnification using the same setting for all images excluding close up images. The ET-DSR settings were the medium gain / exposure used previously. The resulting BF and fluorescent images are shown in FIGS. 14A-14D.

[0310] To quantify migration of the peptides away from the application site, the peptide application site and non-leaf background was identified in the bright field image using color thresholding using FIJI, which created a selection set. This selection set was applied to the 8bit DSR image and the leaf non-application site w as selected by inverting the selection. This area w as measured using the ratio of Integrated Area to Area which yields the average pixel value and normalized by dividing by the mean pixel value of the sample treated with water. In addition, square regions were drawn basal and apical to the application site which span the width of the leaf. These regions w ere drawn such that they began approximately the radius of the application spot away from the application spot to place the region outside of any intense application site signal. The results of the image analysis are shown in are shown in FIGS. 14E-14F.

[0311] Fluorescence was observed and measured both pLAA and ELF (FIGS. 14B-14D). In pLAA, the mean fluorescence outside of the application site correlated to the concentration of peptide applied (FIG. 14E). Leaves treated with ELF, which did not exhibit this relationship (FIG.14E). In addition, water-control-normalized fluorescence analysis indicated observablePATENT APPLICATION DOCKET NO. P14953WOOOfluorescence in regions both apical and basal relative to the application site for the peptides, suggesting that the peptides are mobilized in both the phloem and xylem (FIG. 14F).Conclusion

[0312] Taken together, these results demonstrate penetration and movement within cereal rye leaves for several peptides. Several peptides exhibited strong fluorescence distal to the application site in at least one experiment, with SYS (FIGS. 12C and 13D-13E), THSII (FIGS. 12F and 13G), and pLAA (FIGS. 12H, 13L, and 14C) exhibiting movement across all experiments. As discussed in Examples 4 and 5, these results are surprising given that SYS (tomato) and THSII (tobacco) are not even native to monocots, let alone rye plants. The ability of SYS and THSII to penetrate the epidermis of r e leaves and move within rye plants, as well as the dicots Arabidopsis (Examples 1 and 4) and Palmer amaranth (Example 5), demonstrates the applicability of these peptides for delivering proteins to plants across a wide variety of plant species.

[0313] Movement of each of CEP (FIG. 12B), THSI (FIG. 12E), PEP (FIG. 12G). CpLAA (FIG.121) and ELF (FIGS. 14B and 14D) was observed in one, but not all, experiments. Without wishing to be bound by theory, this variability may be due to the highly hydrophobic waxy cuticle present on monocot leaves, which may make consistent entry7of the peptides a challenge.Example 7: Foliar Spray Application of Proteins

[0314] This Example demonstrates spray application of peptides to leaves of intact rye and Amaranth plants and entry7into vasculature.

[0315] Rye was sown in Berger BM7, using 2.5” pots (32 / flat) at 27°C, and grown under 500pmol photons m'V with a 16 / 8 day night cycle for 11 days. Amaranth was sown in Promix FPX, using 2.5” pots (32 / flat), at24°C and grown under 150 pmol photons m'V under a 16 / 8 day night cycle for 25 days. All plants were fertilized with Jacks General Fertilizer (20-20-20) at label rate whenever watered.

[0316] A stainless steel bench was covered with aluminum foil and plants w ere laid on the surface. Treatment leaves were identified (VI leaf for Rye, best looking ~V4 leaf for Amaranth) and two standard glass slides were placed on top of the treatment leaf, spaced 7mm apart (FIGS. 16A and 17A). Rye leaves were marked with a sharpie at the base of the basal slide to provide rapid identification of the treatment leaf. Treated Amaranth leaves w ere tagged with a thin strip of labPATENT APPLICATION DOCKET NO. PI4953WOOOmarking tape around the petiole. All plants (both Rye and Amaranth) within treatment group were processed together and the aluminum foil covering the table was replaced between treatments.

[0317] An essential oil sprayer was loaded with up to 1 mL of 0.25 mg / rnL peptide treatment solution and one spray was applied to the application leaf of each plant, sprayed from approximately 4-6"’ from the surface of the leaf, such that the spray was applied between the glass slides. The essential oil sprayers emitted 50-70 pL of solution per spray. Each treatment was allowed to rest until visibly dry (—15 minutes), at which point the slides were removed and the plants returned to the Conviron.

[0318] Plants were allowed to grow for three days before imaging.

[0319] All frames were imaged under three channels / exposure: Brightfield, DSR Low (this is the ‘normal’ intensity used ~0.2s exposure), and DSR High (~0.5s exposure). All images were taken with a 0.7x magnification. Leaves were sandwiched between two glass slides. Rye leaves were imaged from the base to tip with ~3 images which are nearly non-overlapping. Amaranth leaves were imaged in different patterns depending on the leaf shape.

[0320] The results for rye are shown in FIGS. 16A-16L and the results for amaranth are shown in FIGS. 17A-17V. In both rye and amaranth, the application site is clearly visible in the fluorescence images of the TAMRA control and the peptide-treated leaves as a rectangle containing fluorescence signal, indicating successful spray ing of the leaf surface exposed between the two glass slides. In the top panels of FIG. 16G, strong fluorescence signal is visible which appears to be due to excess spray of the peptide, rather than uptake and movement of the peptide from the application site.

[0321] In rye, only minor fluorescent signal is visible in the application site in the TAMRA control (FIG. 16C). Plants treated with systemin show some fluorescence associated with vasculature within the application site (FIG. 16E); however, fluorescence extending beyond the application site is likely due to experimental artifacts resulting in leaf damage, as can be seen as localized color changes in the brightfield images (FIG. 16D-16E). In plants treated with pLAA, fluorescence associated with the vasculature is visible within the application site of all replicates, with strong vascular fluorescence visible in replicates 2-5 (FIG. 16G). Some vascular fluorescence appears outside the application site in replicates 1, 2, and 5 treated with pLAA (FIG. 16G), and does not appear to be associated with any visible leaf surface damage as shown in the brightfield imagePATENT APPLICATION DOCKET NO. PI4953WOOO(FIG. 16F). Plants treated with pLAAC showed fluorescence within the application site, with replicates 1, 3, 4, and 5 showing some fluorescence associated with the vasculature, and replicate 5 having some fluorescence associated with vasculature that appears to have moved outside the application site (FIG. 161).

[0322] In amaranth, more fluorescent signal was generally obser ed within the application site of the free TAMRA control as compared to rye. with some fluorescence associated with the vasculature within the application site (FIGS. 17C-17D). The application sites of plants treated with systemin are also clearly visible (FIGS. 17E-17J), with FIG. 17E having visible diffuse fluorescence distal of the application site, and FIGS. 17H and 17J having clear fluorescence associated with the vasculature outside of the application site. Four out of six plants treated with pLAA also had fluorescence associated with the vasculature outside of the application site (FIGS.17K, 17M, 17N, and 17P), with FIGS. 17M and 17P having fluorescence associated with several veins distal of the application site. Three out of four plants treated with pLAAC also also had fluorescence associated with the vasculature outside of the application site (FIGS. 17Q, 17T, and 17U).

[0323] These results indicate that the tested peptides may enter the plant and move within plant vasculature when applied as a foliar spray.

Claims

PATENT APPLICATION DOCKET NO. P14953WOOOWHAT IS CLAIMED IS:

1. A method of delivering a protein into a plant, plant part, or plant cell, the method comprising contacting the plant, plant part, or plant cell with a composition comprising the protein, the protein comprising a polypeptide selected from a plant signaling peptide and an elicitor polypeptide, wherein the polypeptide is from a source organism that is of a different family, genus, and / or species than the plant, plant part, or plant cell that is contacted with the composition, and wherein the protein a) enters plant cells, b) moves within the plant, or c) both (a) and (b).

2. The method of claim 1, wherein the polypeptide is between 10 and 40 amino acids in length.

3. The method of claim 1 or 2, wherein the polypeptide is between 15 and 35 amino acids in length.

4. The method of any one of claims 1-3, wherein at least 2, 3, 4, 5, 6, 7 or 8 of the residues in the polypeptide are lysine, arginine, and / or histidine residues.

5. The method of any one of claims 1-4, wherein at least 10% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues.

6. The method of any one of claims 1-5, wherein between 10% and 40% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues.

7. The method of any one of claims 1-6, wherein between 20% and 30% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues.

8. The method of any one of claims 1-7, wherein at least 3, 4, 5, or 6 of the residues in the polypeptide are proline and / or hydroxyproline residues.

9. The method of any one of claims 1-8, wherein at least 15% of the amino acids in the polypeptide are proline and / or hydroxyproline residues.

10. The method of any one of claims 1-9, wherein between 15% and 50% of the amino acids in the polypeptide are proline and / or hydroxy proline residues.PATENT APPLICATION DOCKET NO. PI4953WOOO11. The method of any one of claims 1-10, wherein the elicitor polypeptide is an ELF 18 polypeptide.

12. The method of any one of claims 1-11, wherein the plant signaling polypeptide is a CEP polypeptide, a HypSys polypeptide, a systemin polypeptide, or a PEP polypeptide.

13. The method of claim 12, wherein the systemin polypeptide comprises an amino acid sequence according to Formula (I): AX1X2SX3PPX4KRX5PPKMQTD. whereinXI is V or A;X2 is R, Q, or H;X3 is T or K;X4 is T or S;and X5 or D or P (SEQ ID NO: 15).

14. The method of claim 13, wherein the systemin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 11-14, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.

15. The method of any one of claims 1-10, wherein the polypeptide is selected from the group consisting of systemin (SEQ ID NO: 4), THSI (SEQ ID NO: 5), THSII (SEQ ID NO: 6), pLAA (SEQ ID NO: 7), pLAAC (SEQ ID NO: 9), CpLAA (SEQ ID NO: 8), CEP1 (SEQ ID NO: 1), PEP1 (SEQ ID NO: 2), and ELF18 (SEQ ID NO: 3), or a polypeptide having at least 60%. 70%.80%, 90%, or 95% sequence identity thereto.

16. A method of delivering a protein into a plant, plant part, or plant cell the method comprising contacting the plant with a composition comprising the protein, the protein comprising a HypSys polypeptide, wherein the protein enters the plant and a) enters a plant cell, b) moves within the plant or plant part, or c) both (a) and (b).

17. A method of delivering a protein into a plant, the method comprising contacting the leaves of the plant with a composition comprising the protein, the protein comprising a polypeptide selected from the group consisting of a HypSys polypeptide, CEP1 (SEQ ID NO: 1), and ELF18 (SEQ ID NO: 3), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto, wherein the protein enters the plant and a) enters a plant cell, b) moves within the plant or plant part, or c) both (a) and (b).PATENT APPLICATION DOCKET NO. P14953WOOO18. The method of claim 12, 16, or 17, wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (III): X1ZX2X3ZX4X5, whereinEach Z is P or O;XI is P, O, S, T, or A;X2 is P, O, S, or T;X3 is P, O, S, T, or A;X4 is K, T, A, or E; andX5 is P, O, S, T, H, K, orY,wherein O is hydroxyproline.

19. The method of claim 12, 16, or 17, wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (IV): X1OOX2OX3, whereinO is hydroxyproline;XI is P, O, S, or A;X2 is O, S, T, or A; andX3 is T, K, A, or E.

20. The method of claim 12, 16, or 17, wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (V): X1PPPX2PX3X4, whereinXI is P, L, or S;X2 is E, S, or A;X3 is K, Q, or E; andX4 is P, K, D, or H(SEQ ID NO:34).

21. The method of claim 12, 16, or 17, wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (II): R(Xl)nX2ZX3X4ZX5X6X7X8X9Xl 0X11X12, whereinn is 4, 5, or 6;each Z is P or O;Each XI is A, D, E, G, H, I, K, L, N, P, R, S, T, V, or Y;X2 is P, O, S, T, or A;X3 is P, O, S, or T;PATENT APPLICATION DOCKET NO. P14953WOOOX4 is P, O, S, T, or A;X5 is K, T, A, or E;X6 is P, O, S, T, H, K, or Y;X7 is S, D, A, E, or Q;X8 is D, P, O, S, I, A, or no amino acid;X9 is P, O, G, E, I, H, S, or no amino acid;XI 0 is S, Q, T, I, Y, K, N, or no amino acid;XI 1 is K. R, H, N, G, or no amino acid; andX12 is Q. P, E. or no amino acid,wherein O is hydroxyproline.

22. The method of claim 12, 16, or 17. wherein the HypSys polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6 and 16-33, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.

23. The method of claim 12, 16, or 17, wherein the HypSys polypeptide is a TobHypSys polypeptide.

24. The method of claim 18, wherein the TobHypSys polypeptide is THSI (SEQ ID NO: 5) or THSII (SEQ ID NO: 6), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.

25. The method of any one of claims 1-24, wherein the protein further comprises a cargo domain.

26. A method of delivering a protein into a plant, the method comprising spraying the leaves of the plant with a composition comprising the protein, the protein comprisinga cargo domain; anda polypeptide configured to enter the leaves of the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b).

27. The method of claim 26, wherein the polypeptide comprises a plant signaling polypeptide and / or an elicitor polypeptide.PATENT APPLICATION DOCKET NO. P14953WOOO28. A method of delivering a protein into a plant, plant part, or plant cell, the method comprising contacting the plant, plant part, or plant cell with a composition comprising the protein, the protein comprisinga cargo domain; anda polypeptide configured to enter the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b),wherein the polypeptide comprises a plant signaling peptide and / or an elicitor polypeptide.

29. The method of any one of claims 25-28, wherein the cargo domain and the polypeptide form a complex through non-covalent interactions.

30. The method of any one of claims 25-28, wherein the cargo domain and the polypeptide are covalently attached, optionally via a peptide bond or a triazole ring.

31. The method of claim 30, wherein the protein comprises a peptide linker between the cargo domain and the polypeptide.

32. A method of delivering a protein into a plant, the method comprising contacting the plant with a composition comprising the protein, the protein comprisinga cargo domain; andpLAA (SEQ ID NO: 7), or a polypeptide having at least 60%. 70%. 80%. 90%. or 95% sequence identity thereto,wherein the cargo domain and pLAA are covalently attached, andwherein the protein enters the plant and a) enters a plant cell, b) moves within the plant, or c) both (a) and (b).

33. The method of claim 32, wherein the cargo domain and the pLAA are covalently attached via a triazole ring.

34. The method of claim 25, wherein the cargo domain and the pLAA are attached via a peptide bond, optionally wherein the protein comprises a peptide linker between the cargo domain and the pLAA.

35. The method of any one of claims 1-16, 18-25, and 28-34, wherein the contacting comprises contacting the leaves of the plant with the composition.PATENT APPLICATION DOCKET NO. P14953WOOO36. The method of claim 35, wherein the contacting the leaves of the plant with the composition comprises applying one or more droplets of the composition onto the leaves of the plant.

37. The method of claim 35 or 36, wherein the contacting the leaves of the plant with the composition comprises spraying the composition onto the leaves of the plant.

38. The method of any one of claims 35-37, wherein the method comprises pre-treating the leaves of the plant with a pretreatment composition comprising a surfactant prior to contacting or spraying the leaves of the plant with the composition comprising the protein.

39. The method of claim 38, wherein the method comprises spraying the leaves of the plant with the pretreatment composition.

40. The method of any one of claims 1-16, 18-25, and 28-34, wherein the contacting comprises applying the composition to the roots or to the soil in which the roots are growing.

41. The method of any one of claims 1-40, wherein the plant is an intact plant.

42. The method of any one of claims 1-41, wherein the plant is present in an agricultural field.

43. The method of any one of claims 1-24 and 35-42, wherein the polypeptide is derived from a source organism that is a different family, genus, and / or species than the species of the plant that is contacted with the composition.

44. The method of claim 43, wherein the source organism is a bacterium.

45. The method of claim 43, wherein the source organism is a source plant of a different family, genus, and / or species than the plant.

46. The method of claim 45, wherein the source plant is a dicot and the plant is a monocot.

47. The method of claim 45 or 46, wherein the source plant is tobacco, tomato, or Arabidopsis thaliana.

48. The method of any one of claims 1-47, wherein the plant is a monocot.PATENT APPLICATION DOCKET NO. P14953WOOO49. The method of claim 48, wherein the plant is rye or maize.

50. The method of any one of claims 1-47, wherein the plant is a dicot.

51. The method of claim 50, wherein the plant is Palmer amaranth.

52. The method of claim 50, wherein the plant is Arabidopsis thaliana.

53. The method of any one of claims 25-52, wherein the cargo domain comprises a plant signaling polypeptide, an antigen-binding polypeptide, a site-directed nuclease, a transcription factor, a pesticidal protein, and / or a protein that enhances a plant trait, optionally wherein the plant trait is yield, drought tolerance, heat tolerance, frost resistance, nutrient use efficiency, abiotic stress tolerance, or a quality trait.

54. The method of any one of claims 1-53, wherein the composition further comprises a surfactant.

55. The method of any one of claims 1-54, wherein the composition further comprises a pesticide.

56. The method of any one of claims 1-55, wherein the composition further comprises glyphosate, glufosinate, 2, 4-di chlorophenoxy acetic acid, and / or dicamba.

57. The method of any one of claims 1-56, wherein the composition is a liquid.

58. The method of any one of claims 1-57, wherein the composition comprises the protein in an amount of at least about 0.01 mg / mL.

59. The method of claim 58, wherein the composition comprises the protein in an amount of at least about 0.10 mg / mL.

60. The method of any one of claims 1-58, wherein the composition comprises the protein in an amount of between 0.01 mg / mL and 10 mg / mL.

61. The method of claim 60, wherein the composition comprises the protein in an amount of between 0.1 mg / mL and 1 mg / mL.PATENT APPLICATION DOCKET NO. P14953WOOO62. The method of claim 61, wherein the composition comprises the protein in an amount of about 0.1 mg / mL, about 0.25 mg / mL, about 0.5 mg / mL, or about 1 mg / mL.

63. An isolated protein comprising a cargo domain and a polypeptide configured to enter the leaves of the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b), wherein the polypeptide comprises a plant signaling or elicitor polypeptide.

64. The protein of claim 63, wherein the polypeptide is between 10 and 40 amino acids in length.

65. The protein of claim 63 or 64, wherein the polypeptide is between 15 and 35 amino acids in length.

66. The protein of any one of claims 63-65, wherein at least 2, 3, 4, 5, 6, 7 or 8 of the residues in the polypeptide are lysine, arginine, and / or histidine residues.

67. The protein of any one of claims 63-66, wherein at least 10% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues.

68. The protein of any one of claims 63-67, wherein between 10% and 40% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues.

69. The protein of any one of claims 63-68, wherein between 20% and 30% of the amino acids in the polypeptide are lysine, arginine, and / or histidine residues.

70. The protein of any one of claims 63-69, wherein at least 3, 4, 5, or 6 of the residues in the polypeptide are proline and / or hydroxyproline residues.

71. The protein of any one of claims 63-70, wherein at least 15% of the amino acids in the polypeptide are proline and / or hydroxyproline residues.

72. The protein of any one of claims 63-71, wherein between 15% and 50% of the amino acids in the polypeptide are proline and / or hydroxyproline residues.

73. The protein of any one of claims 63-72, wherein the plant signaling polypeptide is a CEP polypeptide, a HypSys polypeptide, a systemin polypeptide, or a PEP polypeptide.PATENT APPLICATION DOCKET NO. P14953WOOO74. The protein of claim 73, wherein the systemin polypeptide comprises an amino acid sequence according to Formula (I): AX1X2SX3PPX4KRX5PPKMQTD, whereinXI is V or A;X2 is R, Q, or H;X3 is T or K;X4 is T or S;and X5 or D or P (SEQ ID NO: 15).

75. The protein of claim 74, wherein the systemin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 11-14, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.

76. The protein of claim 73, wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (III): X1ZX2X3ZX4X5, whereinEach Z is P or O;XI is P, O, S, T, or A;X2 is P, O, S, or T;X3 is P, O, S, T, or A;X4 is K, T, A, or E; andX5 is P, O, S, T, H, K, orY,wherein O is hydroxyproline.

77. The protein of claim 73, wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (IV): XI OOX2OX3, whereinO is hydroxyproline;XI is P, O, S, or A;X2 is O, S, T, or A; andX3 is T, K, A, or E.

78. The protein of claim 73, wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (V): X1PPPX2PX3X4, whereinXI is P, L, or S;X2 is E, S, or A;X3 is K, Q, or E; andPATENT APPLICATION DOCKET NO. P14953WOOOX4 is P, K, D, or H(SEQ ID NO:34).

79. The protein of claim 73, wherein the HypSys polypeptide comprises an amino acid sequence according to Formula (II): R(Xl)nX2ZX3X4ZX5X6X7X8X9X10Xl 1X12, wherein n is 4, 5, or 6;each Z is P or O;Each XI is A, D, E, G, H, I, K, L, N, P, R. S, T, V, or Y;X2 is P, O. S, T, or A;X3 is P, O, S, or T;X4 is P, O, S, T, or A;X5 is K, T, A, or E;X6 is P, O, S, T, H, K, or Y;X7 is S, D, A, E, or Q;X8 is D, P, O, S, I, A, or no amino acid;X9 is P, O, G, E, I, H, S, or no amino acid;XI 0 is S, Q, T, I. Y, K. N, or no amino acid;XI 1 is K, R, H, N, G, or no amino acid; andXI 2 is Q, P, E, or no amino acid,wherein O is hydroxyproline.

80. The protein of claim 73, wherein the HypSys polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6 and 16-33, or an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.

81. The protein of any one of claims 63-72, wherein the elicitor polypeptide is an ELF18 polypeptide.

82. The protein of any one of claims 63-74, wherein the polypeptide is selected from the group consisting of a systemin (SEQ ID NO: 4). THSI (SEQ ID NO: 5), THSII (SEQ ID NO: 6), pLAAC (SEQ ID NO: 9), CpLAA (SEQ ID NO: 8), CEP1 (SEQ ID NO: 1), PEP1 (SEQ ID NO: 2), and ELF18 (SEQ ID NO: 3), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity thereto.PATENT APPLICATION DOCKET NO. P14953WOOO83. The protein of any one of claims 63-82, wherein the cargo domain and the polypeptide form a complex through non-covalent interactions.

84. The protein of any one of claims 63-82, wherein the cargo domain and the polypeptide are covalently attached, optionally via a peptide bond or a triazole ring.

85. The protein of claim 84, wherein the protein comprises a peptide linker between the cargo domain and the polypeptide.

86. The protein of any one of claims 63-85, wherein the cargo domain is from a different source organism than the plant signaling or elicitor polypeptide.

87. A recombinant protein comprising a cargo domain and pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAA, wherein the cargo domain and the pLAA are covalently attached.

88. The protein of claim 87, wherein the cargo domain and the pLAA are covalently attached via a triazole ring.

89. The protein of claim 87, wherein the cargo domain and the pLAA are covalently attached via a peptide bond, optionally wherein the protein comprises a peptide linker between the cargo domain and the pLAA.

90. A recombinant protein comprising a polypeptide configured to enter the leaves of the plant and a) enter plant cells, b) move within the plant, or c) both (a) and (b), the polypeptide comprising:CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1), andpLAA (SEQ ID NO: 7), or a polypeptide having at least 60%. 70%. 80%. 90%. or 95% sequence identity7to pLAA,wherein the CEP1 and the pLAA are fused via a peptide bond.

91. The protein of claim 90, wherein the protein comprises a peptide linker between the CEP1 and the pLAA, optionally wherein the peptide linker comprises between 1 and 10 glycine and serine residues.PATENT APPLICATION DOCKET NO. P14953WOOO92. The protein of claim 91, wherein the linker is G4S (SEQ ID NO: 10).

93. The protein of claim 91 or 92, wherein the protein comprises, from N-terminus to C-terminus:CEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1);optionally, the peptide linker; andpLAA (SEQ ID NO: 7), or a polypeptide having at least 60%. 70%. 80%. 90%. or 95% sequence identity to pLAA.

94. The protein of claim 93, wherein the protein comprises CpLAA (SEQ ID NO: 8), or a polypeptide having at least 60%. 70%, 80%, 90%, or 95% sequence identity to CpLAA (SEQ ID NO: 8).

95. The protein of claim 91 or 92, wherein the protein comprises, from N-terminus to C-terminus:pLAA (SEQ ID NO: 7), or a polypeptide having at least 60%. 70%. 80%. 90%. or 95% sequence identity to pLAA;optionally, the peptide linker; andCEP1 (SEQ ID NO: 1), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to CEP1 (SEQ ID NO: 1).

96. The protein of claim 95, wherein the protein comprises is pLAAC (SEQ ID NO: 9), or a polypeptide having at least 60%, 70%, 80%, 90%, or 95% sequence identity to pLAAC (SEQ ID NO: 9).

97. The protein of any one of claims 90-96, further comprises a cargo domain, optionally wherein the cargo domain is fused to the N-terminus or the C-terminus of the polypeptide via a peptide bond.

98. The protein of any one of claims 63-97, wherein the cargo domain comprises a plant signaling polypeptide, an antigen-binding polypeptide, a site-directed nuclease, a transcription factor, a pesticidal protein and / or a protein that enhances a plant trait, optionally wherein thePATENT APPLICATION DOCKET NO. P14953WOOOplant trait is yield, drought tolerance, heat tolerance, frost resistance, nutrient use efficiency, abiotic stress tolerance, or a quality trait.

99. The protein of any one of claims 63-98, wherein the cargo domain is a recombinant or synthetic polypeptide.

100. A composition comprising the protein of any one of claims 63-99 and a suitable carrier.

101. The composition of claim 100, further comprising a surfactant.

102. The composition of claim 100 or 101, further comprising a pesticide.

103. The composition of any one of claims 100-102, wherein the herbicide comprises glyphosate, glufosinate, 2, 4-di chlorophenoxy acetic acid, and / or dicamba.

104. The composition of any one of claims 100-103, wherein the composition is a liquid.

105. The composition of any one of claims 100-104, wherein the composition comprises the protein in an amount of at least about 0.01 mg / mL.

106. The composition of claim 105, wherein the composition comprises the protein in an amount of at least about 0.10 mg / mL.

107. The composition of any one of claims 100-106, wherein the composition comprises the protein in an amount of between 0.01 mg / mL and 10 mg / mL.

108. The composition of claim 107, wherein the composition comprises the protein in an amount of between 0.1 mg / mL and 1 mg / mL.

109. The composition of claim 108, wherein the composition comprises the protein in an amount of about 0.1 mg / mL, about 0.25 mg / mL, about 0.5 mg / mL, or about 1 mg / mL.

110. A plant, plant part, or plant cell comprising the protein of any one of claims 63-99.

111. The plant, plant part, or plant cell of claim 110, wherein the plant is an intact plant.

112. The plant, plant part, or plant cell of claim 111, wherein the intact plant is present in an agricultural field.PATENT APPLICATION DOCKET NO. P14953WOOO113. The plant, plant part, or plant cell of any one of claims 110-112, wherein the polypeptide is derived from a source organism that is a different family, genus, and / or species than the species of the plant, plant part, or plant cell.

114. The plant, plant part, or plant cell of claim 113, wherein the source organism is a bacterium.

115. The plant, plant part, or plant cell of claim 113, wherein the source organism is a source plant of a different family, genus, and / or species than the plant, plant part, or plant cell.

116. The plant, plant part, or plant cell of claim 115, wherein the source plant is a dicot and the plant is a monocot.

117. The plant, plant part, or plant cell of claim 115 or 116, wherein the source plant is tobacco, tomato, or Arabidopsis thaliana.

118. The plant, plant part, or plant cell of any one of claims 110-117, wherein the plant is a monocot.

119. The plant, plant part, or plant cell of claim 118, wherein the plant is rye or maize.

120. The plant, plant part, or plant cell of claim 110-119, wherein the plant is a dicot.

121. The plant, plant part, or plant cell of claim 120, wherein the plant is Palmer amaranth.

122. The plant, plant part, or plant cell of claim 120, wherein the plant is Arabidopsis thaliana.

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