WO2023205876A1

WO2023205876A1 - Antibodies against disease causing agents of plants and uses thereof

Info

Publication number: WO2023205876A1
Application number: PCT/CA2023/050279
Authority: WO
Inventors: Hamlet ABNOUSI; Slade LOUTET; Tsz Ying Sylvia CHEUNG; Audrey TUPIN
Original assignee: Novobind Livestock Therapeutics Inc.
Priority date: 2022-04-29
Filing date: 2023-03-03
Publication date: 2023-11-02

Abstract

The present technology generally relates to methods and antibodies useful for reducing, eliminating, or preventing infection with a bacterial population in a plant.

Description

ANTIBODIES AGAINST DISEASE CAUSING AGENTS OF PLANTS AND USES THEREOF

FIELD OF TECHNOLOGY

[0001 ] The present technology relates to methods and compositions for the control of microorganisms associated with infections in plant xylem and uses thereof.

BACKGROUND

[0002] Economically important plants can suffer from severe disease caused by pathogenic microorganisms that reduces productivity.

[0003] Amongst infectious diseases, the bacterium Xylella fastidiosa is one of the leading vascular pathogens in economically important perennial plants. This bacterium causes such diseases as Pierce’s Disease in grapes, Almond Leaf Scorch in almonds, and Olive Quick Decline Syndrome in olives (Rapicavoli et al, 2018). Bacteria require an insect vector for transport from diseased to healthy plants (Chatterjee et al, 2008). Diseased plants typically show symptoms of scorched leaves and leaf necrosis at the margins (Rapicavoli et al, 2018). Disease control relies on chemical treatment of insect vectors that propagate Xylella fastidiosa between plants (Kyrkou et al, 2018) or the destruction, through uprooting or burning, of diseased plants (Bucci, 2018).

[0004] There is a need for the development of pathogen-specific molecules that inhibit these infections or the association of these pathogens with their hosts. Additionally, there is a need to develop pathogen-specific molecules that can be used to deliver other molecules for pathogen elimination. Finally, there is a need to develop immunomodulatory molecules that can be delivered to plants for.

SUMMARY OF TECHNOLOGY

[0005] With reference to the definitions set out below, described herein are polypeptides comprising heavy chain variable region fragments (VHHS) whose intended use includes but is not limited to the following applications in plant health or an unrelated field: diagnostics, in vitro assays, injectables, formulations, prophylactics, therapeutics, substrate identification, nutritional supplementation, bioscientific and medical research, and companion diagnostics. Also described herein are polypeptides comprising VHHS that bind and decrease the virulence of disease-causing agents in plants. Further to these descriptions, set out below are the uses of polypeptides that comprise VHHS in methods of reducing transmission and severity of disease in host plants, including their use as an ingredient in a product. Further described are the means to produce, characterise, refine, and modify VHHS for this purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 : Shows the structure of the exopolysaccharide (EPS) produced by Xylella fastidiosa (Fastidian Gum) and described in figure 3 of da Silva et al (2001 ).

[0007] FIG. 2: Shows a schematic of camelid heavy chain only antibodies and their relationship to VHH domains and complementarity determining regions (CDRs).

[0008] FIG. 3: Shows phage ELISA binding data for VHH antibodies of this disclosure.

[0009] FIG. 4: Shows functional assays that can be used to assess the activity of VHH antibodies after passage through plants.

[0010] FIG. 5: Shows that NBX0018 (SEQ ID NO: 115) maintains functional activity after passage through the vascular system of olive, calamansi, fig, orange, almond, and grape plants.

[0011] FIG. 6: Shows that NBX18029 (SEQ ID NO: 118), NBX12006 (SEQ ID NO: 116), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding active after passage through the vascular system of olive plants.

[0012] FIG. 7: Shows that NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding active after passage through the vascular system of grape and almond plants.

[0013] FIG. 8: Shows that NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding activity after passage through the vascular system of orange plants.

[0014] FIG. 9: Shows that NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding activity after passage through the vascular system of fig plants. [0015] FIG. 10: Shows that NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding activity after passage through the vascular system of calamansi plants.

[0016] FIG. 11 : Shows the presence of NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) in grape and almond plant extracts and the presence of NBX0018 (SEQ ID NO: 115) in almond, grape, and orange plant extracts.

DEFINITIONS

[0017] In describing the present technology, the following terminology is used in accordance with the definitions below.

[0018] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

Host

[0019] As referred to herein, “host”, “host organism”, “recipient plant”, “host plant”, “recipient animal”, “host animal” and variations thereof refer to the intended recipient of the product when the product constitutes a prophylactic or a therapeutic. In certain embodiments, the host is from the order Vitales. In certain embodiments, the host is from the family Vitaceae. In certain embodiments, the host is from the genus Vitis. In certain embodiments, the host is a grape plant. In certain embodiments, the host is from the order Rosales. In certain embodiments, the host is from the family Rosaceae. In certain embodiments, the host is from the genus Prunus. In certain embodiments, the host is an almond, plum or peach plant. In certain embodiments, the host is from the genus Ficus. In certain embodiments, the host is a fig plant. In certain embodiments, the host is from the order Lamiales. In certain embodiments, the host is from the family Oleaceae. In certain embodiments, the host is from the genus Olea. In certain embodiments, the host is an olive plant. In certain embodiments, the host is from the order Sapindales. In certain embodiments, the host is from the family Rutaceae. In certain embodiments, the host is from the genus Citrus. In certain embodiments, the host is a citrus plant. In certain embodiments, the host is from the order Fabales. In certain embodiments, the host is from the family Fabaceae. In certain embodiments, the host is from the genus Medicago. In certain embodiments, the host is an alfalfa plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Vinca. In certain embodiments, the host is a periwinkle plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Rubiaceae. In certain embodiments, the host is from the genus Coffea. In certain embodiments, the host is a coffee plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Nerium. In certain embodiments, the host is an oleander plant. In certain embodiments, the host is from the order Fagales. In certain embodiments, the host is from the family Juglandaceae. In certain embodiments, the host is from the genus Carya. In certain embodiments, the host is a pecan plant. In certain embodiments, the host is from the order Ericales. In certain embodiments, the host is from the family Ericaceae. In certain embodiments, the host is from the genus Vaccinium. In certain embodiments, the host is a berry-producing plant. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Cicadellidae. In certain embodiments, the host is a leafhopper. In certain embodiments, the host is a sharpshooter. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Aphrophoridae. In certain embodiments, the host is a spittlebug.

Pathogens [0020] As referred to herein, “pathogen”, “pathogenic”, and variations thereof refer to virulent microorganisms, that can be associated with host organisms, that give rise to a symptom or set of symptoms in that organism that are not present in uninfected host organisms, including the reduction in ability to survive, thrive, or reproduce. Without limitation, pathogens encompass parasites, bacteria, viruses, prions, protists, fungi, and algae. In certain embodiments, the pathogen is a bacterium belonging to the order Xanthomonadales. In certain embodiments, the pathogen is a bacterium belonging to the family Xanthomonadaceae. In certain embodiments, the pathogen is a bacterium belonging to the genus Xylella. In certain embodiments, the pathogen is Xylella fastidiosa. In certain embodiments, the pathogen is Xylella fastidiosa subspecies fastidiosa. In certain embodiments, the pathogen is Xylella fastidiosa subspecies multiplex. In certain embodiments, the pathogen is Xylella fastidiosa subspecies pauca. [0021] “Virulence”, “virulent” and variations thereof refer to a pathogen’s ability to cause symptoms in a host organism. “Virulence factor” refers to nucleic acids, plasmids, genomic islands, genes, peptides, proteins, toxins, lipids, macromolecular machineries, or complexes thereof that have a demonstrated or putative role in infection.

[0022] “Disease-causing agent” refers to a microorganism, pathogen, or virulence factor with a demonstrated or putative role in infection.

Bacteria

[0023] As referred to herein, “bacteria”, “bacterial” and variations thereof refer, without limitation, to bacteria of the Xylella genus, or any other bacterial species associated with host organisms. In certain embodiments, a bacterium may not be virulent in all host organisms it is associated with.

Exopolysaccharide (EPS)

[0024] As referred to herein, “exopolysaccharide”, “EPS”, “fastidian gum”, “gum” and variations thereof refer, without limitation, to the sugar-containing, high molecular weight polymers synthesized by Xylella fastidosa, or any other bacteria.

Antibodies

[0025] A schematic of camelid heavy chain only antibodies and their relationship to VHH domains and complementarity determining regions (CDRs) is shown in FIG. 2. (Panel A) A camelid heavy chain only antibody consists of two heavy chains linked by a disulphide bridge. Each heavy chain contains two constant immunoglobulin domains (CH2 and CH3) linked through a hinge region to a variable immunoglobulin domain (VHH). (Panel B) Each VHH domain contains an amino acid sequence of approximately 110-130 amino acids. The VHH domain consists of the following regions starting at the N-terminus (N): framework region 1 (FR1 ), complementarity-determining region 1 (CDR1 ), framework region 2 (FR2), complementarity-determining region 2 (CDR2), framework region 3 (FR3), complementarity-determining region 3 (CDR3), and framework region 4 (FR4). The domain ends at the C-terminus (C). The complementarity-determining regions are highly variable, determine antigen binding by the antibody, and are held together in a scaffold by the framework regions of the VHH domain. The framework regions consist of more conserved amino acid sequences; however, some variability exists in these regions.

[0026] As referred to herein “VHH” refers to an antibody or antibody fragment comprising a single heavy chain variable region which may be derived from natural or synthetic sources. NBXs referred to herein are an example of a VHH. In a certain aspect, a VHH may lack a portion of a heavy chain constant region (CH2 or CH3), or an entire heavy chain constant region.

[0027] As referred to herein “heavy chain antibody” refers to an antibody that comprises two heavy chains and lacks the two light chains normally found in a conventional antibody. The heavy chain antibody may originate from a species of the Camelidae family or Chondrichthyes class. Heavy chain antibodies retain specific binding to an antigen in the absence of any light chain.

[0028] As referred to herein “specific binding”, “specifically binds” or variations thereof refer to binding that occurs between an antibody and its target molecule that is mediated by at least one complementarity determining region (CDR) of the antibody’s variable region. Binding that is between the constant region and another molecule, such as Protein A or G, for example, does not constitute specific binding.

[0029] As referred to herein “antibody fragment” refers to any portion of a conventional or heavy chain antibody that retains a capacity to specifically bind a target antigen and may include a single chain antibody, a variable region fragment of a heavy chain antibody, a nanobody, a polypeptide or an immunoglobulin new antigen receptor (IgNAR).

[0030] As referred to herein an “antibody originates from a species” when any of the CDR regions of the antibody were raised in an animal of said species. Antibodies that are raised in a certain species and then optimized by an in vitro method (e.g., phage display) are considered to have originated from that species.

[0031] As referred to herein “conventional antibody” refers to any full-sized immunoglobulin that comprises two heavy chain molecules and two light chain molecules joined together by a disulfide bond. In certain embodiments, the antibodies, compositions, therapeutic formulations, prophylactic formulations, products, and methods described herein do not utilize conventional antibodies.

Production System

[0032] As referred to herein, “production system” and variations thereof refer to any system that can be used to produce any physical embodiment of the technology or modified forms of the technology. Without limitation, this includes but is not limited to biological production by any of the following: bacteria, yeast, algae, arthropods, arthropod cells, plants, mammalian cells. Without limitation, biological production can give rise to antibodies that can be intracellular, periplasmic, membrane-associated, secreted, or phage-associated. Without limitation, “production system” and variations thereof also include, without limitation, any synthetic production system. This includes, without limitation, de novo protein synthesis, protein synthesis in the presence of cell extracts, protein synthesis in the presence of purified enzymes, and any other alternative protein synthesis system.

Product

[0033] As referred to herein, “product” refers to any physical embodiment of the technology or modified forms of the technology, wherein the binding of the VHH to any molecule, including itself, defines its use. Without limitation, this includes a formulation, a formulation additive, a nutritional supplement, a premix, a medicine, a prophylactic, a spray, a therapeutic, an injectable, a drug, a diagnostic tool, a component or entirety of an in vitro assay, a component or the entirety of a diagnostic assay (including companion diagnostic assays), a component of a delivery system, or an immunomodulatory molecule

Formulation product

[0034] As referred to herein, “formulation product’’ refers to any physical embodiment of the technology or modified forms of the technology, wherein the binding of the VHH to any molecule, including itself, defines its intended use as a product that is taken up by a host organism. Without limitation, this includes a formulation, a formulation additive, a nutritional supplement, a premix, a medicine, a prophylactic, a therapeutic, a spray, an injectable, or a drug.

DETAILED DESCRIPTION OF TECHNOLOGY

[0035] Descriptions of the technology provided are to be interpreted in conjunction with the definitions and caveats provided herein.

[0036] Hundreds of plant species are grown around the world for food production. Many pathogenic micro-organisms lead to plant food production losses. Amongst economically important perennial plant species, vascular pathogens include bacteria, such as members of the Xylella genus and fungi. From this group, one organism of particular interest is the bacterium Xylella fastidiosa which causes Pierce’s Disease in grapes, Almond Leaf Scorch in almonds, and Olive Quick Decline Syndrome in olives (Rapicavoli et al, 2018). Together, these diseases cause annual losses in the 100s of millions of dollars to producers. Other plant diseases caused by Xylella fastidiosa include citrus variegated chlorosis, alfalfa dwarf, periwinkle wilt, peach phony disease and leaf scorch in olives, plums, coffee, oleander, pecan, and vaccinium berry plants.

[0037] The lifecycle of Xylella fastidiosa within the plant is a complex one (Bucci, 2018). Initially, after Xylella fastidiosa enters a plant xylem, planktonic cells utilize the xylem to spread within the plant. Next the bacteria adhere to host cells and form bacterial cell aggregates known as biofilm. As bacterial numbers increase, they begin producing enzymes that degrade host cell walls and expand pores between xylem vessels, further allowing the bacteria to spread through the plant. Diseased plants exhibit such symptoms as leaf scorching, necrosis at leaf margins, and berry desiccation (Rapicavoli et al, 2018). To spread between plants, Xylella fastidiosa requires an insect vector. Xylem sap- feeding insects, such as leafhoppers and sharpshooters, ingest bacteria from the xylem of an infected plant and then directly inoculate the xylem of healthy plants with the bacteria (Chatterjee et al, 2008).

[0038] Current Xylella fastidiosa control measures include the application of chemicals that target the insect vectors; however, these compounds can harm helpful insects such as bees and there is growing insect resistance to these compounds (Kyrkou et al, 2018). Alternatively, farmers can uproot or burn diseased plants; however, this is a costly control mechanism, and it takes many years before replanted perennial plants produce significant new crops. This also requires co-ordination and co-operation between different farmers (Bucci, 2018). Since plants do not possess an adaptive immune system (Andolfo and Ercolano, 2015), the development of a Xylella fastidiosa vaccine is not possible.

[0039] Secreted Xylella fastidiosa proteins that have been implicated in nutrient acquisition or host cell wall degradation include ChiA (Labroussaa et al, 2017), LesA (Nascimento et al, 2016), and PglA (Roper et al, 2007). The type 1 fimbriae (Li et al, 2007; Feil et al, 2007) and the autotransporter XatA (Matsumoto et al, 2012) of Xylella fastidiosa are both involved in virulence through roles in cell-cell aggregation and biofilm formation. Additionally, exopolysaccharide (EPS) produced by Xylella fastidiosa contributes to virulence by facilitating bacterial adhesion to surfaces and biofilm formation (Killiny et al, 2013). Finally, the outer membrane protein TolC of Xylella fastidiosa is likely playing a role in virulence through both the secretion of enzymes necessary for virulence and the removal of toxic phytochemicals from the bacteria (Reddy etal, 2007). Genetic disruption of any of the systems described above gives rise to Xylella fastidiosa isolates with significantly decreased capacity to cause disease in plants. Some of these virulence factors, including ChiA (Labroussaa et al, 2017), PglA (Killiny and Almeida, 2014), EPS (Killiny et al, 2013), also contribute to vector transmission of Xylella fastidiosa between plants.

[0040] Earlier efforts in the field of this technology rely on the chemical control of leafhopper and sharpshooter insects that serve as vectors for the transport of Xylella fastidiosa between plants (Kyrkou et al, 2018). To date, vector control has been hindered by a lack of chemicals targeting all vector species and environmental contamination caused by chemical control affecting other insects, such as bees, and the development of insect resistance to the chemical controls (Kyrkou et al, 2018). The effectiveness of prior arts is limited by these challenges. These problems are circumvented by introducing exogenous peptides that neutralise the virulence and spread of the disease-causing agent into the host via injection without eliciting the host immune response. Moreover, the methods described herein provide scope for the adaptation and refinement of neutralising peptides, which provides synthetic functionality beyond what the host is naturally able to produce.

[0041] Antibody heavy chain variable region fragments (VHHS) are small (12-15 kDa) proteins that comprise specific binding regions to antigens. When introduced into a plant, VHHS bind and neutralise the effect of disease-causing agents in situ. Owing to their smaller mass, they are less susceptible than conventional antibodies, such as previously documented IgYs, to cleavage by enzymes found in host organisms, more resilient to temperature and pH changes, more soluble, have low systemic absorption and are easier to recombinantly produce on a large scale, making them more suitable for use in plant therapeutics than conventional antibodies. Furthermore, HHS introduced into plants can be used to deliver other molecules for specific pathogen elimination. When introduced into plants, VHHS may also modulate plant immunity.

Antibodies for preventing or reducing virulence (summary)

[0042] In one aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents to reduce the severity and transmission of disease between and across species. In certain embodiments, the VHH is supplied to host plants. In certain embodiments, the VHH is an ingredient of a product.

Binding to reduce virulence

[0043] In another aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents, and in doing so, reduce the ability of the disease-causing agent to exert a pathological function or contribute to a disease phenotype. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the rate of replication of the disease-causing agent. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to bind to its cognate receptor. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to degrade components of the host organism. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to interact with another molecule or molecules. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to reach the site of infection. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to cause cell death.

Binding to deliver other molecules

[0044] In another aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and can be bound to another molecule. In certain embodiments, the VHH or VHHS deliver the other molecule to a specific pathogen. In certain embodiments, the other molecule can eliminate the specific pathogen targeted by the VHH or VHHS.

Antibodies derived from llamas

[0045] In a further aspect, the present technology provides a method for the inoculation of camelid or other species with recombinant virulence factors, the retrieval of mRNA encoding VHH domains from lymphocytes of the inoculated organism, the reverse transcription of mRNA encoding VHH domains to produce cDNA, the cloning of cDNA into a suitable vector and the recombinant expression of the VHH from the vector. In certain embodiments, the camelid can be a dromedary, camel, llama, alpaca, vicuna, or guanaco, without limitation. In certain embodiments, the inoculated species can be, without limitation, any organism that can produce single domain antibodies, including cartilaginous fish, such as a member of the Chondrichthyes class of organisms, which includes for example sharks, rays, skates and sawfish. In certain embodiments, the heavy chain antibody comprises a sequence set forth in Table 1 . In certain embodiments, the heavy chain antibody comprises an amino acid sequence with at least 80%, 90%, 95%, 97%, or 99% identity to any sequence disclosed in Table 1. In certain embodiments, the heavy chain antibody possesses a CDR1 set forth in Table 2. In certain embodiments, the heavy chain antibody possesses a CDR2 set forth in Table 2. In certain embodiments, the heavy chain antibody possesses a CDR3 set forth in Table 2.

Antibodies recombinantly expressed

[0046] In another aspect, the present technology provides a method for producing VHH in a suitable producing organism. Suitable producing organisms include, without limitation, bacteria, yeast, and algae. In certain embodiments, the producing bacterium is Escherichia coli. In certain embodiments, the producing bacterium is a member of the Bacillus genus. In certain embodiments, the producing bacterium is a probiotic. In certain embodiments, the yeast is Pichia pastoris. In certain embodiments, the yeast is Saccharomyces cerevisiae. In certain embodiments, the yeast is Yarrowia lipolytica. In certain embodiments, the alga is a member of the Chlamydomonas or Phaeodactylum genera.

Antibodies as a therapeutic

[0047] In yet another aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or HHS that bind disease-causing agents and are administered to host plants via any suitable route as a therapeutic. In certain embodiments, the plant is selected from the list of host plant described, with that list being representative but not limiting. In certain embodiments, the route of administration to a recipient plant can be, but is not limited to: inclusion in any part of the plant, introduction to the xylem, introduction to the phloem, provided to the exterior surface (for example, as a spray or submersion), provided to the medium in which the plant dwells, provided by injection, provided via diffusion, provided via absorption by roots, provided genetically as a genetically-modified plant, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects. In certain embodiments, the route of administration to a recipient insect can be but is not limited to: inclusion in any part of the insect, provided by insect feed, provided by insect traps, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects. In certain embodiments, the VHH or VHHS deliver a secondary molecule to the pathogen. In certain embodiments, the VHH or VHHS modulate the immune system of the host. In certain embodiments, the host is from the order Vitales. In certain embodiments, the host is from the family Vitaceae. In certain embodiments, the host is from the genus Vitis. In certain embodiments, the host is a grape plant. In certain embodiments, the host is from the order Rosales. In certain embodiments, the host is from the family Rosaceae. In certain embodiments, the host is from the genus Prunus. In certain embodiments, the host is an almond, plum or peach plant. In certain embodiments, the host is from the genus Ficus. In certain embodiments, the host is a fig plant. In certain embodiments, the host is from the order Lamiales. In certain embodiments, the host is from the family Oleaceae. In certain embodiments, the host is from the genus Olea. In certain embodiments, the host is an olive plant. In certain embodiments, the host is from the order Sapindales. In certain embodiments, the host is from the family Rutaceae. In certain embodiments, the host is from the genus Citrus. In certain embodiments, the host is a citrus plant. In certain embodiments, the host is from the order Fabales. In certain embodiments, the host is from the family Fabaceae. In certain embodiments, the host is from the genus Medicago. In certain embodiments, the host is an alfalfa plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Vinca. In certain embodiments, the host is a periwinkle plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Rubiaceae. In certain embodiments, the host is from the genus Coffea. In certain embodiments, the host is a coffee plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Nerium. In certain embodiments, the host is an oleander plant. In certain embodiments, the host is from the order Fagales. In certain embodiments, the host is from the family Juglandaceae. In certain embodiments, the host is from the genus Carya. In certain embodiments, the host is a pecan plant. In certain embodiments, the host is from the order Ericales. In certain embodiments, the host is from the family Ericaceae. In certain embodiments, the host is from the genus Vaccinium. In certain embodiments, the host is a berry-producing plant. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Cicadellidae. In certain embodiments, the host is a leafhopper. In certain embodiments, the host is a sharpshooter. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Aphrophoridae. In certain embodiments, the host is a spittlebug.

Therapeutic dosage

[0048] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents to host plants as part of a product at any suitable dosage regime. In practice, the suitable dosage is the dosage at which the product offers any degree of protection against a diseasecausing agent, and depends on the delivery method, delivery schedule, the environment of the recipient plant, the size of the recipient plant, the age of the recipient plant and the health condition of the recipient plant among other factors. In certain embodiments, VHHS are administered to recipient plants at a concentration in excess of 1 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration in excess of 5 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration in excess of 10 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration in excess of 50 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration in excess of 100 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration less than 1 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration less than 500 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration less than 100 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration less than 50 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration less than 10 mg/kg of plant weight. In certain embodiments, VHHS are administered to fields at a concentration in excess of 1 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration in excess of 5 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration in excess of 10 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration in excess of 50 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration in excess of 100 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration less than 1 kg/ha.

Therapeutic frequency

[0049] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and are administered to host plants as part of a product at any suitable dosage frequency. In practice, the suitable dosage frequency is that at which the product offers any protection against a disease-causing agent, and depends on the delivery method, delivery schedule, the environment of the recipient plant, the size of the recipient plant, the age of the recipient plant and the health condition of the recipient plant, among other factors. In certain embodiments, the dosage frequency can be but is not limited to: constantly, at consistent specified frequencies under an hour, hourly, at specified frequencies throughout a 24-hour cycle, daily, at specified frequencies throughout a week, weekly, at specified frequencies throughout a month, monthly, at specified frequencies throughout a year, annually, and at any other specified frequency greater than 1 year.

Antibodies added to a prophylactic formulation

[0050] In yet another aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and are administered to host plants via any suitable route as part of a prophylactic or therapeutic product. In certain embodiments, the plant is selected from the list of host plants described, with that list being representative but not limiting. In certain embodiments, the route of administration to a recipient plant can be, but is not limited to: inclusion in any part of the plant, introduction to the xylem, introduction to the phloem, provided to the exterior surface (for example, as a spray or submersion), provided to the medium in which the plant dwells, provided by injection, provided via diffusion, provided via absorption by roots, provided genetically as a genetically-modified plant, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects. In certain embodiments, the route of administration to a recipient insect can be, but is not limited to: inclusion in any part of the insect, provided by insect feed, provided by insect traps, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects. In certain embodiments, the host is from the order Vitales. In certain embodiments, the host is from the family Vitaceae. In certain embodiments, the host is from the genus Vitis. In certain embodiments, the host is a grape plant. In certain embodiments, the host is from the order Rosales. In certain embodiments, the host is from the family Rosaceae. In certain embodiments, the host is from the genus Prunus. In certain embodiments, the host is an almond, plum or peach plant. In certain embodiments, the host is from the genus Ficus. In certain embodiments, the host is a fig plant. In certain embodiments, the host is from the order Lamiales. In certain embodiments, the host is from the family Oleaceae. In certain embodiments, the host is from the genus Olea. In certain embodiments, the host is an olive plant. In certain embodiments, the host is from the order Sapindales. In certain embodiments, the host is from the family Rutaceae. In certain embodiments, the host is from the genus Citrus. In certain embodiments, the host is a citrus plant. In certain embodiments, the host is from the order Fabales. In certain embodiments, the host is from the family Fabaceae. In certain embodiments, the host is from the genus Medicago. In certain embodiments, the host is an alfalfa plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Vinca. In certain embodiments, the host is a periwinkle plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Rubiaceae. In certain embodiments, the host is from the genus Coffea. In certain embodiments, the host is a coffee plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Nerium. In certain embodiments, the host is an oleander plant. In certain embodiments, the host is from the order Fagales. In certain embodiments, the host is from the family Juglandaceae. In certain embodiments, the host is from the genus Carya. In certain embodiments, the host is a pecan plant In certain embodiments, the host is from the order Ericales. In certain embodiments, the host is from the family Ericaceae. In certain embodiments, the host is from the genus Vaccinium. In certain embodiments, the host is a berry-producing plant. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Cicadellidae. In certain embodiments, the host is a leafhopper. In certain embodiments, the host is a sharpshooter. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Aphrophoridae. In certain embodiments, the host is a spittlebug.

Prophylactic formulation product

[0051] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or HHS that bind disease-causing agents and are administered to host plants in the form of a product. The form of the product is not limited. In certain embodiments, the product is a formulation, nutritional supplement, premix, prophylactic, therapeutic, spray, injectable, medicine, formulation additive, or feed but is not limited to these forms.

Formulation additives

[0052] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or HHS that bind disease-causing agents and are administered to host plants as part of a product that also comprises other additives or coatings. In practice, the most suitable coating or additive depends on the method of delivery, the recipient plant, the environment of the recipient, the nutritional requirements of the recipient plant, the frequency of delivery, the age of the recipient plant, the size of the recipient plant, or the health condition of the recipient plant. In certain embodiments, these additives and coatings can include but are not limited to the following list and mixtures thereof: a vitamin, an amino acid, a dye, an antibiotic, an antiviral, a hormone, an antimicrobial peptide, a steroid, a prebiotic, a probiotic, a bacteriophage, chitin, chitosan, B-1 ,3-glucan, vegetable extracts, peptone, an additive spray, a toxin binder, a yeast extract, a plant extract, sugar, a detoxifying enzyme, an enzyme inhibitor, an essential mineral, carnitine, glucosamine, an essential salt, a preservative, a stabilizer, a pesticide, a herbicide, or water.

Non-prophylactic and non-therapeutic uses

[0053] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and can be used in a non-prophylactic or non-therapeutic use, such as but not limited to: a diagnostic kit, an enzyme-linked immunoabsorbent assay (ELISA), a western blot assay, an immunofluorescence assay, or a fluorescence resonance energy transfer (FRET) assay, in its current form and/or as a polypeptide conjugated to another molecule. In certain embodiments, the conjugated molecule can be but is not limited to: a fluorophore, a chemiluminescent substrate, an antimicrobial peptide, a nucleic acid, or a lipid.

Antigens

[0054] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH orVnHs that bind disease-causing agents, produced by a bacterium of the order Xanthomonadales. In certain embodiments, the Xanthomonadales bacterium refers to both current and reclassified bacteria. In certain embodiments, the pathogen is a bacterium belonging to the Xanthomonadaceae family. In certain embodiments, the pathogen is a bacterium belonging to the genus Xylella. In certain embodiments, the pathogen is Xylella fastidiosa.

[0055] In certain embodiments, the VHH or plurality thereof is capable of binding to one or more disease-causing agents, originating from the same or different bacteria. In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein ChiA (SEQ ID NO: 109). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein LesA (SEQ ID NO: 110). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein PglA (SEQ ID NO: 111 ). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein FimF (SEQ ID NO: 112). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein XatA (SEQ ID NO: 113). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein TolC (SEQ ID NO: 114). In certain embodiments, the disease-causing agent is the exopolysaccharide produced by Xylella fastidiosa (FIG. 1 ). In certain embodiments, the preferred antigens are ChiA (SEQ ID NO: 109), LesA (SEQ ID NO: 10), and PglA (SEQ ID NO: 111 ) as their recombinant expression and purification is anticipated to be simpler than other antigens. In certain embodiments, the disease-causing agent is a molecule secreted by Xylella fastidiosa. In certain embodiments, the disease-causing agent is a surface associated molecule of Xylella fastidiosa. In certain embodiments, the disease-causing agent is a molecule in the outer membrane of Xylella fastidiosa.

Antigen Sequences

[0056] Xylella fastidiosa protein ChiA (SEQ ID NO: 109)

[0057] >AAO29659.1 conserved hypothetical protein [Xylella fastidiosa

Temeculal]

[0058] MTQRPFALLLTLIFALPATAKNPTALFYLMNSDKSTNSFLAHADKIDTLVP

TWYRVDPTGWNGTPNEHVYRIAQQKKITLTPILSMSGSRDGFHTLLHDEAAKTRMNT FLVKESKARGYKGFQFDFENIAQTDRGAYTLMVKQTAEALHKAGMTLSIAIVPNAPGH PEEGGDFSKWMWEYWRGVYDLKALGQAADLISLMTYDQHTRWTTPGPVAGMPWT KKHLEYALTQVPKEKLSLGIPTYGYRWFTGNPVRKDGTENSNISGTYIDADKSFPLAIA QKANVQWDPIEQESWFYFYRDNMREWVFRPDAHSFRARYDLVKQNGLQGFSCWVL GAEDPKMWDELPLATH

[0059] Xylella fastidiosa protein LesA (SEQ ID NO: 110)

[0060] >AAO29541.1 conserved hypothetical protein [Xylella fastidiosa

Temeculal]

[0061] MVINRKIIGSYTQRQIAALLTDEPPSEQPKCNVRWEMTYTTVGWGEPA

RASGWLVPDGPQCPGPYPVLGWGSATETLRSAEQAKGIISANGDDPLVTRLASQGY WVSTDYLGLGGSNYPYHPYLHANSEASALIDALRASRTVLQHLNTPLSNKLMLSGFS QGGHTAMATQREIEAHLSNEFSLVASAPISGPYALSQTFLDTWTGRNEVGENNFAIIL GTYAIVGMQRTYRNIYSDPSQVFQDPWANQVEALFPGKKDTTDLFLGDDLPAIDQIKE YFQPGFYSDFPKNRKNPFFVDLVRNDLLNWAPRTPTLLCGSDNDTWPFLKNTNLAIA

SFKKRGSRQVSWDIGTGNRKDNSALTHLSSEELCIVKVRNEFLEKKR

[0062] Xylella fastidiosa protein PglA (SEQ ID NO: 111 )

[0063] >AAO29329.1 polygalacturonase precursor [Xylella fastidiosa Temeculal]

[0064] MNLDRFLPLVFGLHCFAAMGGTADVFRVSQLSSSDLSEPVSVKTLWGE

VQRPSLPTHVCTVLPARLTPKHGSIDALDANPKVSKPDTKRLQDAIDDCPAGSAVKLVI

DSHRKSGFLSGPLHLKSGVTLWIDEGVTLFASRNPKDYDKGNGTCGTATSTHEFSCM

PLISAINTTGSGIVGGGVIDGRGGSILTGGKYARQRTWWDLAYQNKRHALHQQVPRLI

QIRGGNDFTLYRVAIENAPNFHVVADTVSGVTAWGIRILTPSLVYTTPGYHCPPGTTPD

WTPATCFTPDTVKNTDGFDPGQSNHVLLAYSYISTGDDHVAIKARGKMPSYALSFLH

NHFGYGHGMSIGSDTESGVHDMEVSDLSIDGFDSPNSNGLQMKSDADHGGWDHVT

YSKICMRRLKRPLAFDTFYKPSNGNSYPLFKNIVLQDIHVLESPVFGAGQLLFMGILGS

GNNLPMTLSMDNVVFDGFLPTLIAPPSSWFANPQAVHFHFGPGPVSFAPLITPSVAY

DVTVSGSPGVGNPYDCSAAFINFSSVFPDSPI

[0065] Xylella fastidiosa protein FimF (SEQ ID NO: 112)

[0066] >AAO27959.1 fimbrial adhesin precursor [Xylella fastidiosa Temeculal ]

[0067] MSIVNTISVAHISTYQQRTLKIIAACLTMLIAPHVQATCTIKEGIVAQNIDFG

AGRILIQPSLAIGDRIALLTHNINRVDRYGTCVNNGIMYGAFTRSMTPVSGMKNVYATD

VAGVGIRLYREAGKVSNFYPHTLNSNNPPGRVTNLSLNEGYFQVELIKTATTTGSGPIA

SAGPFTTYYADGSGISRPILTSSLSGIGITIVTSTCQVDAGSKNIAVNFGSVSSNTFKGL

GSKGPERDFTINLICHGGNVTEADQAQISVRIDGTQDSSGQSGVLAITSASDAASNIGI

ELVDLLTGSERQVVFGQAITLGRTAINASSTLSLPMRARYIQTQTGKVGPGAAKGTAT FTIEYQ

[0068] Xylella fastidiosa protein XatA (SEQ ID NO: 113)

[0069] >AAO28401.1 conserved hypothetical protein [Xylella fastidiosa

Temeculal]

[0070] MERKNHKKTTLATLISVLLMGSAGATYANSPSISEKLDLATQHLDGAAST

GEAISSDGKTIATSINNRTRAFLLSGDNWATKTELGSLKSDSSGTSIVRALSADGKIAA GISSVDSSSTQLHATIWSGDNWATKTTLGTLATNNGGDSWYALSADGKVAAGQTAA SIGTLHAVIWSGDNWATKTDLGTIRSDGTGDSMVKALSADGKIAVGSAASDIKTPVNG MYAGGVLTGPNSVRAIAWSGDNWATKTDLGTLRSDNLGAAVANAISADGKVIGGASH HYVTTEYYRANADQLATIWSGGNWATKTNLGSLRSDNSGASLVAALSADGSIAGGAA

LNDANATRAIIWSGSHWATKTDLGTLKSDNSGNSEWALSPDGSVAVGTAATDSGSS HATVWKITYPTESTSTSDSTTNTQTPSQPSQPSWTVDADNTKKTLSQLGAETFSVM DLQRQGLTRLQQHCQIDAAGQSCWGIQTAISSIDGNRDKVAGIRLGHGFTETFSAGVS LDRSLSRSLPDSYLKNNGNLGAGLYAQWNTAFKGSQWYVRPAVAFNRYNVTVQRPV

LENTEAGIGTSRMKGRGASLEAGQTFKSDHGVLLGWHLGGRYNNVSRAEYSEQNAA FPTTYGKASFKRTSAYLGADATIPLTTNLKWMAAVEIDRALKNRDAVYSAQADYIGSF NHRAGIKGTIATLSSGLQYTISEKILLGLTLDVNQTAFGDIARGGTFTVGGHF

[0071 ] Xylella fastidiosa protein TolC (SEQ ID NO: 114)

[0072] >AAO29793.1 outer membrane export factor [Xylella fastidiosa Temeculal ]

[0073] MIRRCLALAVTTALFPVVSQATNLLQVYEMARAADPQLSAAESTRLYSVE GQVQARAALLPQMSGTATLARSRTEYDGISGSAISKNRQYSINGSQTLFNWSQFSNL

RSQREIAKAADFTLESAKQNLIVRSSAAYFSVLIGIESLIAAEANEASAKRQFDYAQKRL EVGLSPITDLHEARASYDQARADTITARNTLQDYYQALAEITGQPVTDLRGLPEDFRPE VPAKFSNIDQLVTEAISNNPSLKAQQLQVNAAESSISAARGEHYPTLSLSGSLGKSKA WGSSSGAMSALIPDRNQPISDTNNLQLTLSIPLFAGGATQSRVRQAIAQRDIQQDNYE

QNKRTLNRNTRNAYQNMIAGISEVEARRLAVVSAQAALDASQVGLEVGTRTLLDVVQ NQRILFSVRQAYVQARYNFLQNRLLLSQNSGTLSIEDVQEINQLLTADSERKL

EXAMPLES

[0074] The following illustrative examples are representative of the embodiments of the applications, systems and methods described herein and are not meant to be limiting in any way.

[0075] While preferred embodiments of the present technology are shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the technology. It should be understood that various alternatives to the embodiments of the technology described herein may be employed in practicing the technology.

Production of antigens

[0076] Recombinant antigens can be purified from an E. coli expression system. For example, a 6xHis-tagged ChiA was expressed at 30°C in E. coli BL21 (DE3) cells grown overnight in LB media with 0.5 mM IPTG induction at an optical density at 600 nM of 0.8. Cells were then lysed by sonication in buffer A (250 mM NaCI, 50 mM CaCL, 20 mM Imidazole and 10 mM HEPES, pH 7.5) with 12.5 pg/ml DNase I, and 1 mM PMSF. The lysate was cleared by centrifugation at 22000 x g for 30 minutes at 4°C, applied to a 5 ml HisTrap HP column (GE Healthcare) pre-equilibrated with buffer A, washed with ten column volumes of buffer A and eluted with a gradient of 0% to 60% (vol/vol) buffer B (250 mM NaCI, 500 mM Imidazole and 10 mM HEPES, pH 7.5). The protein was then dialyzed overnight into buffer C (10 mM Tris, pH 8.8, 5 mM NaCI) at 4°C. The dialyzed protein was applied to a HiTrap HP ion exchange column (GE Biosciences) preequilibrated with buffer C. The protein was eluted with a gradient of 0% to 50% (vol/vol) buffer D (10 mM Tris, pH 8.8, 1 .0 M NaCI). Lastly, the eluate was loaded onto a Sephacryl S100 gel filtration column (GE Healthcare) using PBS buffer. The protein sample was then concentrated to 1 mg/mL using Am icon concentrators with appropriate molecular weight cut-off (MWCO; Millipore). The purified protein was stored at -80°C.

Production of NBXs and panning

Llama immunization

[0077] A single llama was immunized with purified disease-causing agents, such as the antigens listed, which may be accompanied by adjuvants. The llama immunization was performed using 100 pg of each antigen that were pooled and injected. At the time of injection, the antigens were thawed, and the volume increased to 1 ml with PBS. The 1 ml antigen-PBS mixture was then mixed with 1 ml of Complete Freund’s adjuvant (CFA) or Incomplete Freund’s adjuvant (IFA) for a total of 2 ml. A total of 2 ml was immunized per injection. The immunization was done five times (days 0, 21 , 42, 63, and 70). Whole llama blood and sera were collected from the immunized animal on days 0, 28, 49, 84. Sera from days 28, 49 and 84 were fractionated to separate VHH from conventional antibodies. ELISA were be used to measure reactivity against target antigens in polyclonal and VHH-enriched fractions. Lymphocytes were collected from sera taken at days 28, 49, and 84.

Panning

[0078] RNA isolated from purified llama lymphocytes was used to generate cDNA for cloning into phagemids. The resulting phagemids were used to transform E. coh TG- 1 cells to generate a library of expressed VHH genes. The phagemid library size was ~2.5 x 10⁷ total transformants and the estimated number of phagemid containing VHH inserts was >90%. High affinity antibodies were then selected by panning against the antigens used for llama immunization. Three rounds of panning were performed and antigen-binding clones arising from round 3 were identified using phage ELISA. Antigenbinding clones were sequenced, grouped according to their CDR sequences, and prioritized for soluble expression in E. coh and antibody purification.

[0079] FIG. 3 shows the phage ELISA results for antibodies of this disclosure. Black bars show binding to wells coated with the antigen specified in Tables 1 and 2 dissolved in phosphate-buffered saline (PBS). Grey bars are negative controls that show binding to wells coated with PBS only. In all cases binding to the antigen target is at least four times above binding to the PBS-coated wells.

Purification of VHHS from E. coh

[0080] TEV protease-cleavable, 6xHis-thioredoxin-NBX fusion proteins were expressed in the cytoplasm of E. coh grown in autoinducing media (Formedium) for 24 hours at 30°C. Bacteria were collected by centrifugation, resuspended in buffer A (10 mM HEPES, pH 7.5, 250 mM NaCI, 20 mM Imidazole) and lysed using sonication. Insoluble material was removed by centrifugation and the remaining soluble fraction was applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer A. The protein is eluted from the column using an FPLC with a linear gradient between buffer A and buffer B (10 mM HEPES, pH 7.5, 250 mM NaCI, 500 mM Imidazole). The eluted protein was dialyzed overnight in the presence of TEV protease to buffer C (10 mM HEPES, pH 7.5, 250 mM NaCI). The dialyzed protein was applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer C. 6xHis-tagged TEV and 6xHis-tagged thioredoxin were bound to the column and highly purified NBX was collected in the flow through. NBX proteins were further purified using ion-exchange chromatography. NBX 1 proteins were dialyzed overnight to 20 mM HEPES, pH7.4, 150 mM NaCI and concentrated to ~10 mg/ml.

Purification of VHHS from P. pastoris

[0081] Pichia pastoris strain GS115 with constructs for the expression and secretion of 6xHis-tagged VHH were grown for 5 days at 30°C with daily induction of 0.5% (vol/vol) methanol. Yeast cells were removed by centrifugation and the NBX- containing supernatant was spiked with 10 mM imidazole. The supernatant was applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer A (10 mM HEPES, pH 7.5, 250 mM NaCI). The protein was eluted from the column using an FPLC with a linear gradient between buffer A and buffer B (10 mM HEPES, pH 7.5, 250 mM NaCI, 500 mM Imidazole). NBX proteins were further purified using ion-exchange chromatography. NBX proteins were dialyzed overnight to 20 mM HEPES, pH7.4, 150 mM NaCI and concentrated to ~10 mg/ml.

Flow of NBXs through the vascular system of plants

[0082] These examples demonstrate that NBXs can be introduced into the vascular system of a plant and flow to other portions of the plant, where they can be recovered and their activity is maintained.

[0083] NBXs selected for use in these experiments bind to epitopes of antigens of Salmonella enterica serovar Typhimurium. Salmonella bacteria can enter plants through roots, stomata, hydathodes, or at points of plant damage and colonize plant tissues, including edible tissues (Karmakar et al, 2018). This allows Salmonella to cause food- borne infections in humans. In the presence of plant pathogenic bacteria, Salmonella may benefit and increase in numbers within plant tissues (George et al, 2018). As such, existing NBXs targeting Salmonella antigens serve as a good surrogate for the study of the flow of NBXs through plant tissues. The selected NBXs were NBX0018 (SEQ ID NO: 115) which binds Salmonella enterica serovar Typhimurium FliC (SEQ ID NO: 120), NBX12006 (SEQ ID NO: 116) and NBX18029 (SEQ ID NO: 118) which bind Salmonella ente ca serovar Typhimurium StdD (SEQ ID NO: 121 ), and NBX15016 (SEQ ID NO: 117) which binds Salmonella enterica serovar Typhimurium SipD (SEQ ID NO: 122). One other NBX selected for these studies, NBX0880 (SEQ ID NO: 119), binds to Clostridium perfringens NetB (SEQ ID NO: 123). NBX0018 and NBX0880 have previously been described in WO2017199094A1 and W02020035741A2, respectively, which are incorporated in their entirety by reference herein.

[0084] >NBX0018 (SEQ ID NO: 115)

[0085] QVKLEESGGGLVQPGGSLEVSCAASGIIFSPNAMGWYRQAPGEQRELV

ATITSFGIINYADSVKDRFTISRDNAKNTVYLQMTSLKPEDTAVYYCNAKTFDGTRWHD YWGQGTQVTVSS

[0086] >NBX12006 (SEQ ID NO: 116)

[0087] QVQLQESGGGWVQPGGSLRLSCAASGRTFSSYGLGWFRQAPGKERE

FVAAISWSGGSAYYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCAADEIDGL

GNHDFTSWGQGMQVTVSS

[0088] >NBX15016 (SEQ ID NO: 117)

[0089] QVQLQESGGGLVQPGGSLRLSCAASGSIDRAYHMGWYRQAPGKQREL

VATISDGGVTNYGDSAKGRFTITRNGAENTVNLQMNSLKFEDTAVYYCYVLGRSVYW GQGTQVTVSS

[0090] >NBX18029 (SEQ ID NO: 118)

[0091] QVQLQESGGGLVQAGGSLRLSCVASGSIPSINAMVWYRQAPGKEREM

VAGITSGGRTDYADSWGRFTITRDNAKNTVNLQMNSLKPEDTAVYYCAAMYGSTWD RIWGQGTQVTVSS

[0092] >NBX0880 (SEQ ID NO: 119)

[0093] QVQLQESGGGLVQPGGSLRLSCAASVSSIGTMGWFRQAPGKQPELVA

SISRVGTTNYANSVKGRFTVSRDNAQNTMYLQMNSLKPEDTAVYLCFANVISGPVYW GQGTQVTVSS

[0094] Salmonella enterica serovar Typhimurium FliC (SEQ ID NO: 120)

[0095] >sp|P06179|FLIC_SALTY Flagellin OS=Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) GN=fliC PE=1 SV=4 [0096] MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAI

ANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSD

LDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTL

GLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDT

TGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQ

VANADLTEAKAALTAAGVTGTASWKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKD

GSISINTTKYTADDGTSKTALNKLGGADGKTEWSIGGKTYAASKAEGHNFKAQPDLA

EAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDS

DYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR

[0097] Salmonella enterica serovar Typhimurium StdD (SEQ ID NO: 121 )

[0098] >CBW19104.1 probable fimbrial membrane protein [Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344]

[0099] MTEWIFNLKTKLTVLVMMLCSLCVTKVYAVELGINECAVTSGQNINLRSIN

LTTDDFKPGPDSVIYTINHDAVFKCYMGYDTQFPQLVFNQGYFSKFTKTLDAMGLGFR

MSIQETGNASSWSFSWDEIKSTQSGNELRKEFGTKLPVGTTERKVRITLDFLYTKAY

SESSAVTAFTGISNVLNIVPFSYSLRQNGFVLSGFNVRILRNGLGKVDIVPLQVNFGHIY

TTYEPSQTRQANFTVIARQVLRPAMGQEFTIPLAITFGKGALTQDTGQTLNLVSLDGP

NKGQPNGLRLSIKDDKGKEITFDKQEVLGDITITGAVTGNVSKVYTAVITPTPGGSVKT GTFSAAIPVTVTYN

[0100] Salmonella enterica serovar Typhimurium SipD (SEQ ID NO: 122)

[0101] >WP_000932249.1 MULTISPECIES: SPI-1 type III secretion system needle tip complex protein SipD [Salmonella]

[0102] MLNIQNYSASPHPGIVAERPQTPSASEHVETAWPSTTEHRGTDIISLSQ

AATKIQQAQQTLQSTPPISEENNDERTLARQQLTSSLNALAKSGVSLSAEQNENLRSA

FSAPTSALFSASPMAQPRTTISDAEIWDMVSQNISAIGDSYLGVYENVVAVYTDFYQA

FSDILSKMGGWLLPGKDGNTVKLDVTSLKNDLNSLVNKYNQINSNTVLFPAQSGSGV

KVATEAEARQWLSELNLPNSCLKSYGSGYWTVDLTPLQKMVQDIDGLGAPGKDSKL

EMDNAKYQAWQSGFKAQEENMKTTLQTLTQKYSNANSLYDNLVKVLSSTISSSLETA KSFLQG [0103] Clostridium perfringens NetB (SEQ ID NO: 123)

[0104] >ABW71 134.1 necrotic enteritis toxin B precursor [Clostridium perfringens] [0105] MKRLKIISITLVLTSVISTSLFSTQTQVFASELNDINKIELKNLSGEIIKENGK

EAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTALLNLEGFIPSDKQIFGSKYYGKMK WPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINAS YNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRL YNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVIIVEYQRFDNDYILNWETT QWRGTNKLSSTSEYNEFMFKINWQDHKIEYYL

[0106] Many of the selected NBXs can block the function of the virulence factor they bind in vitro. This can allow for the assessment of NBX function after collection from a plant and is in addition to measuring the binding of each NBX to its target antigen.

[0107] NBX0018 (SEQ ID NO: 115) reduces the motility of Salmonella enterica serovar Typhimurium strain SL1344 (FIG. 4A) in an experiment conducted as follows. Overnight cultures of Salmonella enterica serovar Typhimurium strain SL1344 and a non-motile Dflif mutant were used to inoculate subcultures which were grown at 37°C in a shaking incubator until logarithmic growth was reached. Five pl of log phase bacteria were mixed with 10 pl of NBX0018 or PBS. Mixtures were incubated at room temperature for 1 hour and observed at 400x magnification with an Olympus IX70 inverted microscope with an Olympus DP80 camera. Salmonella motility was tracked and analyzed using Velocity image analysis software. The non-motile Dflif mutant shows low overall motility, while the wild-type bacterium has high overall motility in the presence of PBS. NBX0018 reduces the motility of the wild-type strain in a dose-dependent fashion. Each point on the graph indicates the average speed of a tracked bacterium, with hundreds of bacteria tracked for each mixture. The lines indicate the average movement speed of each mixture.

[0108] NBX15016 (SEQ ID NO: 117) reduces the invasiveness of Salmonella enterica serovar Typhimurium into tissue-cultured HeLa cells (FIG. 4B) in an experiment conducted as follows. Overnight cultures of Salmonella enterica serovar Typhimurium strain SL1344 and a non-invasive DsipD mutant were used to inoculate subcultures which were grown at 37°C in a shaking incubator until logarithmic growth was reached. Bacterial cultures were incubated for 30 minutes with NBX15016 or PBS and then the mixtures were applied to HeLa cells for one hour. Non-invaded bacterial cells were removed through washing and treatment with gentamicin. After an additional hour, HeLa cells were lysed and intracellular bacteria were enumerated using colony counting. The number of invasive bacteria was highest with the wild-type bacterium (SL1344). Invasiveness of SL1344 was reduced by ~75 % in the presence of NBX15016. Finally, the non-invasive DsipD mutant had greatly reduced invasiveness, as expected. Each point on the graph indicates the number of intracellular bacteria from a well, and the lines indicate the average number of intracellular bacteria per well.

[0109] NBX0880 (SEQ ID NO: 119) prevents the NetB toxin (SEQ ID NO: 123) from killing tissue-cultured LMH cells (FIG. 4C) in an experiment conducted as follows. Purified NetB protein was mixed with NBX0880 at a range of concentrations for 30 minutes and then the mixtures were applied to LMH cells for five hours. After five hours, lysis of the LMH cells was assessed using a commercial LDH cytotoxicity kit (ProMega). Lysis by NetB in the presence of NBX0880 was scaled relative to the amount of NetB- induced lysis in the absence of NBX0880. As indicated in the figure, NBX0880 reduces NetB toxicity in a dose-dependent fashion.

[0110] In the first example of NBX movement through plants, 30 mM NBX0018 (SEQ ID NO: 115) diluted in PBS was injected into the stem of an olive plant (Olea europaea). The NBX solution was injected at a rate of 0.4 mL/hour for 5.5 hours. After injection, water droplets from leaves were collected (referred to as leaf sweat below). The leaf sweat was applied to a Salmonella enterica motility assay as follows. Overnight cultures of Salmonella enterica serovar Typhimurium strain SL1344 and a non-motile Dflif mutant were used to inoculate subcultures which were grown at 37°C in a shaking incubator until logarithmic growth was reached. Five pl of log phase bacteria were mixed with 10 pl of plant samples or control solutions. Mixtures were incubated at room temperature for 1 hour and observed at 400x magnification with an Olympus IX70 inverted microscope with an Olympus DP80 camera. Salmonella motility was tracked and analyzed using Velocity image analysis software. [0111] Data for this experiment are shown in FIG. 5A. Each point on the graph indicates the average speed of a tracked bacterium, with hundreds of bacteria tracked for each mixture. The lines indicate the average movement speed of each mixture. From left to right the mixtures are as follows. One, the non-motile D/7/7 mutant mixed with PBS, the results for which are expected with very low average motility. Two, the wild-type bacteria mixed with PBS, the results for which are expected with high average motility. Three, the wild-type bacteria mixed with purified NBX0018 at a final concentration of 50 mM, the results for which are expected with motility inhibited by NBX0018. Four, the wildtype bacteria mixed with leaf sweat from an olive plant injected with PBS. The results indicated that there were no naturally-occurring compounds released from the leaves that inhibit bacterial motility. Five, the wild-type bacteria mixed with leaf sweat from an olive plant injected with NBX0018. The results indicate that NBX0018 travels through the plant and maintains its function sufficiently well to inhibit Salmonella motility in a functional in vitro assay.

[0112] Similar results (FIG. 5B) were obtained when NBX0018 was injected into calamansi (Citrus x microcarpa), fig (Ficus carica), and orange (Citrus x sinensis) plants and collected from leaf water droplets. Similar results (FIG. 5B) were also obtained when NBX0018 was injected into the stems of almond (Prunus amygdalus) and grape (Vitis vinifera) plants and material was collected from the opposite end of the stem.

[0113] In the next example of NBX movement through plants, 30 mM NBX18029 (SEQ ID NO: 118), 30 mM NBX12006 (SEQ ID NO: 116), 30 mM NBX0880 (SEQ ID NO: 119), and 30 mM NBX15016 (SEQ ID NO: 117) diluted in PBS were individually injected into olive plants (Olea europaea). The NBX solutions were injected at a rate of 0.4 mL/hour for 6 hours. After injection, water droplets from leaves were collected (referred to as leaf sweat below) and leaves were collected and ground in an extraction buffer (20 mM Tris-HCI pH7.4, 150 mM NaCI, 2.6 mM KOI, 2% PVP40, 0.05% Tween- 20) at a ratio of 1 g of leaves to 5 ml of buffer (referred to as leaf extract below). The leaf extracts were clarified by centrifugation and the supernatants were used in the ELISA as follows. ELISA plate was coated with 100 ml of 1 -10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST). 100 ml of each leaf sweat sample, leaf extract sample or standard curve sample was added to ELISA plate and detected with 1 :5000 dilution of an anti-VHH-HRP antibody (GenScript, A02014-200). 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.

[0114] Data for this example are shown in FIG. 6. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ), panel C, which shows the binding of NBX12006 (SEQ ID NO: 116) to StdD (SEQ ID NO: 121 ), panel E, which shows the binding of NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), and panel F, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122). Panel B shows that after injection of NBX18029 (SEQ ID NO: 118) into an olive plant and collection of leaf sweat and leaf extract, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag). Panel D shows that after injection of NBX12006 (SEQ ID NO: 116) into an olive plant and collection of leaf sweat and leaf extract, NBX12006 (SEQ ID NO: 116) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag). Panel F shows that after injection of NBX0880 (SEQ ID NO: 119) into an olive plant and collection of leaf sweat and leaf extract, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag). Panel G shows that after injection of NBX15016 (SEQ ID NO: 117) into an olive plant and collection of leaf sweat and leaf extract, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag). In the case of each NBX and corresponding antigen (panels B, D, F, and H), there is no significant binding from leaf extracts or sweat from plants injected with PBS alone on antigen coated wells, indicating that the measured binding in the samples injected with NBXs was dependent on the retention of NBX activity after transfer through the olive plant.

[0115] In the next example of NBX movement through plants, a mixture of 30 mM NBX18029 (SEQ ID NO: 118), 30 mM NBX0880 (SEQ ID NO: 119), and 30 mM NBX15016 (SEQ ID NO: 117) diluted in PBS was injected into the stems of almond (Prunus amygdalus) and grape (Vitis vinifera) plants. Material was collected from the opposite end of the stem. These injections were done with a syringe by hand until 0.5 mL of material was collected. The material was then subjected to an ELISA-based binding assay as follows. ELISA plate was coated with 100 ml of 1-10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST). 100 ml of each plant sample or standard curve sample was added to ELISA plate and detected with 1 :5000 dilution of an anti-VHH-HRP antibody (GenScript, A02014-200). 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.

[0116] Data for this example are shown in FIG. 7. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ), panel C, which shows the binding of NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), and panel E, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122). Panel B shows that after injection of the NBX mixture to both almond (Prunus amygdalus) and grape (Vitis vinifera) stems and collection of material from the opposite end of the stems, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag). Panel D shows that after injection of the NBX mixture to both almonds and grape stems and collection of material from the opposite end of the stems, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag). Panel F shows that after injection of the NBX mixture to both almonds and grape stems and collection of material from the opposite end of the stems, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag). In the case of each NBX and corresponding antigen (panels B, D, and F), there is no significant binding from stems injected with PBS alone on antigen coated wells, indicating that the measured binding in the samples injected with NBXs was dependent on the retention of NBX activity after transfer through the stem of the almond or grape plants.

[0117] In the next example of NBX movement through plants, a mixture of 30 mM NBX18029 (SEQ ID NO: 118), 30 mM NBX0880 (SEQ ID NO: 119), and 30 mM NBX15016 (SEQ ID NO: 117) diluted in PBS was injected into an orange plant (Citrus x sinensis'). The NBX solution was injected at a rate of 0.4 mL/hour for 6 hours. After injection, water droplets from leaves were collected (referred to as leaf sweat below) and leaves were collected and ground in an extraction buffer (20 mM Tris-HCI pH7.4, 150 mM NaCI, 2.6 mM KCI, 2% PVP40, 0.05% Tween-20) at a ratio of 1 g of leaves to 5 ml of buffer (referred to as leaf extract below). The leaf extracts were clarified by centrifugation and the supernatants were used in the ELISA as follows. ELISA plate was coated with 100 ml of 1 -10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST). 100 ml of each leaf sweat sample, leaf extract sample or standard curve sample was added to ELISA plate and detected with 1 .5000 dilution of an anti-VHH-HRP antibody (GenScript, A02014-200). 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.

[0118] Data for this example are shown in FIG. 8. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ), panel C, which shows the binding of NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), and panel E, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122). Panel B shows that after injection of NBX18029 (SEQ ID NO: 118) into an orange plant and collection of leaf sweat and leaf extract, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag). Panel D shows that after injection of NBX0880 (SEQ ID NO: 119) into an orange plant and collection of leaf sweat and leaf extract, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag). Panel F shows that after injection of NBX15016 (SEQ ID NO: 117) into an orange plant and collection of leaf sweat and leaf extract, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag). In the case of each NBX and corresponding antigen (panels B, D, and F), there is significantly less binding from leaf extracts or sweat from plants injected with PBS alone on antigen coated wells or uncoated wells, indicating that the measured binding in the samples injected with NBXs was dependent on the retention of NBX activity after transfer through the orange plant.

[0119] In the next example of NBX movement through plants, a mixture of 30 mM NBX18029 (SEQ ID NO: 118), 30 mM NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) diluted in PBS was injected into a fig plant (Ficus carica). The NBX solution was injected at a rate of 0.4 mL/hour for 6 hours. After injection, water droplets from leaves were collected (referred to as leaf sweat below) and leaves were collected and ground in an extraction buffer (20 mM Tris-HCI pH7.4, 150 mM NaCI, 2.6 mM KOI, 2% PVP40, 0.05% Tween-20) at a ratio of 1 g of leaves to 5 ml of buffer (referred to as leaf extract below). The leaf extracts were clarified by centrifugation and the supernatants were used in the ELISA as follows. ELISA plate was coated with 100 ml of 1-10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST). 100 ml of each leaf sweat sample, leaf extract sample or standard curve sample was added to ELISA plate and detected with 1 :5000 dilution of an anti- VHH-HRP antibody (GenScript, A02014-200). 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.

[0120] Data for this example are shown in FIG. 9. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ), panel C, which shows the binding of NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), and panel E, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122). Panel B shows that after injection of NBX18029 (SEQ ID NO: 118) into a fig plant and collection of leaf sweat and leaf extract, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag). Panel D shows that after injection of NBX0880 (SEQ ID NO: 119) into a fig plant and collection of leaf sweat and leaf extract, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag). Panel F shows that after injection of NBX15016 (SEQ ID NO: 117) into a fig plant and collection of leaf sweat and leaf extract, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag). In the case of each NBX and corresponding antigen (panels B, D, and F), there is no significant binding from leaf sweat or leaf extract from a plant injected with PBS alone on antigen coated wells or uncoated wells, indicating that the measured binding in the samples injected with NBXs was dependent on the retention of NBX activity after transfer through the fig plant.

[0121] In the next example of NBX movement through plants, a mixture of 30 mM NBX0880 (SEQ ID NO: 119), 30 mM NBX15016 (SEQ ID NO: 117), and 30 mM NBX18029 (SEQ ID NO: 118) diluted in PBS was injected into a calamansi plant (Citrus x microcarpa). The NBX solution was injected at a rate of 0.4 mL/hour for 6 hours. After injection, water droplets from leaves were collected (referred to as leaf sweat below) and leaves were collected and ground in an extraction buffer (20 mM Tris-HCI pH7.4, 150 mM NaCI, 2.6 mM KCI, 2% PVP40, 0.05% Tween-20) at a ratio of 1 g of leaves to 5 ml of buffer (referred to as leaf extract below). The leaf extracts were clarified by centrifugation and the supernatants were used in the ELISA as follows. ELISA plate was coated with 100 ml of 1 -10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST). 100 ml of each leaf sweat sample, leaf extract sample or standard curve sample was added to ELISA plate and detected with 1 :5000 dilution of an anti-VHH-HRP antibody (GenScript, A02014-200). 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.

[0122] Data for this example are shown in FIG. 10. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), panel C, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122), and panel E, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ). Panel B shows that after injection of NBX0880 (SEQ ID NO: 119) into a calamansi plant and collection of leaf sweat and leaf extract, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag). Panel D shows that after injection of NBX15016 (SEQ ID NO: 117) into a calamansi plant and collection of leaf sweat and leaf extract, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag). Panel F shows that after injection of NBX18029 (SEQ ID NO: 118) into a calamansi plant and collection of leaf sweat and leaf extract, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag). In the case of each NBX and corresponding antigen (panels B, D, and F), there is significantly less binding from leaf extracts or sweat from plants injected with PBS alone on antigen coated wells or uncoated wells, indicating that the measured binding in the samples injected with NBXs was dependent on the retention of NBX activity after transfer through the calamansi plant.

[0123] In the next example, the presence of NBXs in plant derived materials was also visualized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (FIG. 11 ).

[0124] FIG. 11 Panel A demonstrates the transfer of a mixture of NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) through the stems of almond (Prunus amygdalus) and grape (Vitis vinifera) plants. Lane 1 : NBX mixture diluted in PBS prior to injection. Lane 2: Stem extract of grape plant after injection with PBS. Lane 3: Stem extract of grape plant after injection with NBX mixture. Lane 4: Stem extract of almond plant after injection with PBS. Lane 5: Stem extract of almond plant after injection with NBX mixture. Lane 6: Molecular weight marker.

[0125] FIG. 11 Panel B demonstrates the transfer of NBX0018 (SEQ ID NO: 115) through the stems of almond (Prunus amygdalus) and grape (Vitis vinifera) plants and to the leaves of an orange (Citrus x sinensis) plant. Lane 1 : Leaf sweat of orange plant after injection of NBX0018. Lane 2: Leaf sweat of orange plant after injection of PBS. Lane 3: Stem extract of almond plant after injection with NBX0018. Lane 4: Stem extract of almond plant after injection with PBS. Lane 5: Stem extract of grape plant after injection with NBX0018. Lane 6: Stem extract of grape plant after injection with PBS. Lane 7: NBX0018 solution prior to injection. Lane 8: Molecular weight marker.

[0126] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document is specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

The following references are incorporated by reference in their entirety.

[0127] Andolfo, G. & Ercalano, M R. (2015). Plant innate immunity multicomponent model. Frontiers in Plant Science. Volume 6, Article 987.

[0128] Bucci, E.M. (2018). Xylella fastidiosa, a new plant pathogen that threatens global farming: ecology, molecular biology, search for remedies. Biochemical and Biophysical Research Communications. Volume 502, pp. 173-182.

[0129] Chatterjee, S. et al. (2008). Living in two worlds: the plant and insect lifestyles of Xylella fastidiosa. Annual Review of Phytopathology. Volume 46, pp. 243- 271.

[0130] da Silva, F.R. et al. (2001 ). Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity. FEMS Microbiology Letters. Volume 203, pp. 165-171.

[0131] George, A.S. et al. (2018). Interactions of Salmonella enterica serovar Typhimurium and Pecto bacterium carotovorum within a tomato soft rot. Applied and Environmental Microbiology. Volume 84, Article e01913-17.

[0132] Feil, H. et al. (2007). Contribution of fimbrial and afimbrial adhesins of Xylella fastidiosa to attachment to surfaces and virulence to grape. Phytopathology. Volume 97, pp. 318-324.

[0133] Karmakar, K. et al. (2018). Root mediated uptake of Salmonella is different from phyto-pathogen and associated with the colonization of edible organs. BMC Plant Biology. Volume 18, Article 344.

[0134] Ki I liny, N. et al. (2013). The exopolysaccharide of Xylella fastidiosa is essential for biofilm formation, plant virulence, and vector transmission. Molecular Plant- Microbe Interactions. Volume 26, pp. 1044-1053.

[0135] Killiny, N. & Almeida, R.P.P. (2014). Factors affecting the initial adhesion and retention of the plant pathogen Xylella fastidiosa in the foregut of an insect vector. Applied and Environmental Microbiology. Volume 80, pp. 420-426. [0136] Kyrkou, I. et al. (2018). Pierce’s disease of grapevines: a review of control strategies and an outline of an epidemiological model. Frontiers in Microbiology. Volume 9, Article 2141.

[0137] Labroussaa, F. et al. (2017). A chitinase is required for Xylella fastidiosa colonization of its insect and plant hosts. Microbiology. Volume 163, pp. 502-509.

[0138] Li, Y. et al. (2007). Type I and type IV pili of Xylella fastidiosa affect twitching motility, biofilm formation and cell-cell aggregation. Microbiology. Volume 153, pp. 719- 726.

[0139] Matsumoto, A. etal. (2012). XatA, an AT-1 autotransporter important for the virulence of Xylella fastidiosa Temeculal. MicrobiologyOpen. Volume 1 , pp. 33-45.

[0140] Nascimento, R. et al. (2016). The type II secreted lipase/esterase LesA is a key virulence factor required for Xylella fastidiosa pathogenesis in grapevines. Scientific Reports. Volume 6, Article 18598.

[0141] Rapicavoli, J. et al. (2018). Xylella fastidiosa: an examination of a re- emerging plant pathogen. Molecular Plant Pathology. Volume 19, pp. 786-800.

[0142] Reddy, J.D. et al. (2007). TolC is required for pathogenicity of Xylella fastidiosa in Vitis vinifera grapevines. Molecular Plant-Microbe Interactions. Volume 20, pp. 403-410.

[0143] Roper, M.C. et al. (2007). Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines. Molecular Plant-Microbe Interactions. Volume 20, pp. 411-419.

Claims

1. A polypeptide comprising at least one variable region fragment of a heavy chain antibody (VHH), wherein at least one VHH specifically binds a disease-causing agent.

2. The polypeptide of claim 1 , wherein the polypeptide comprises a plurality of VHHS.

3. The polypeptide of claim 2, wherein the polypeptide comprises at least three VHHS.

4. The polypeptide of claim 2 or 3, wherein any one of the plurality of HHS is identical to another VHH of the plurality of VHHS.

5. The polypeptide of any one of claims 2 to 4, wherein the plurality of VHHS are covalently coupled to one another by a linker, the linker comprising one or more amino acids.

6. The polypeptide of any one of claims 1 to 5, wherein the variable region fragment of the heavy chain antibody comprises an amino acid sequence at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: Nos: 1 to 27

7. The polypeptide of any one of claims 1 to 10, wherein the variable region fragment of the heavy chain antibody comprises a complementarity determining region 1 (CDR1 ) as set forth in any one of SEQ ID NO: Nos: 28 to 54, a complementarity determining region 2 (CDR2) as set forth in any one of SEQ ID NO: Nos: 55 to 81 and a complementarity determining region 3 (CDR3) as set forth in any one of SEQ ID NO: Nos: 82 to 108.

8. A polypeptide complex, wherein the polypeptide comprises a first component polypeptide and a second component polypeptide, wherein the first component polypeptide and the second component polypeptide are not covalently linked together and are coupled together by a protein-protein interaction, a small molecule-protein interaction, or a small molecule-small molecule interaction, wherein each of the first and the second component polypeptides comprise a VHH which specifically bind a pathogen.

9. The polypeptide of any one of claims 1 to 7 or the polypeptide complex of claim 8, wherein the pathogen is a plant-associated bacterium.

10. The polypeptide of any one of claims 1 to 7 or the polypeptide complex of claim 8, wherein the plant-associated bacterium is a Gram-negative bacterium.

11 . The polypeptide of any one of claims 1 to 7 or the polypeptide complex of claim 8, wherein the Gram-negative bacterium is from the Xanthomonadales order.

12. The polypeptide of any one of claims 1 to 7 or the polypeptide complex of claim 8, wherein the plant-associated bacterium is Xylella fastidiosa.

13. The polypeptide or the polypeptide complex of claim 12, wherein the VHH specifically binds a Xylella fastidiosa virulence factor.

14. The polypeptide or the polypeptide complex of claim 12, wherein the VHH specifically binds an antigen or polypeptide at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:s Nos: 109 to 114 or combinations thereof.

15. The polypeptide or the polypeptide complex of claim 12, wherein the Xylella fastidiosa virulence factor is ChiA polypeptide, ChiA-like polypeptide, LesA polypeptide, LesA-like polypeptide, PglA polypeptide, PglA-like polypeptide, FimF polypeptide, FimF- like polypeptide, XatA polypeptide, XatA-like polypepide, TolC polypeptide, TolC-like polypeptide.

16. The polypeptide or the polypeptide complex of claim 12, wherein the Xylella fastidiosa virulence factor of claim 13 is ChiA polypeptide, LesA polypeptide, PglA polypeptide.

17. The polypeptide or the polypeptide complex of claim 12, wherein the Xylella fastidiosa virulence factor is Fastidian Gum exopolysaccharide or a Fastidian Gum-like exopolysaccharide.

18. The polypeptide or the polypeptide complex of claim 12, wherein the VHH specifically binds a molecule secreted by Xylella fastidiosa.

19. The polypeptide or the polypeptide complex of claim 12, wherein the VHH specifically binds a surface-associated molecule of Xylella fastidiosa.

20. The polypeptide or the polypeptide complex of claim 12, wherein the VHH specifically binds an outer membrane molecule of Xylella fastidiosa.

21. The polypeptide or the polypeptide complex of claim 12, wherein the VHH specifically binds a molecule of Xylella fastidiosa involved in host toxicity, host cell wall degradation, motility, autoaggregation, biofilm formation, adhesion, or transmission to or from vector.

22. A nucleic acid encoding the polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 .

23. A plurality of nucleic acids encoding the polypeptide complex of any one of claims 8 to 21.

24. A vector comprising the nucleic acid of claim 22 or the plurality of nucleic acids of claim 23 that can be used to produce the polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 in any production system.

25. A cell comprising the nucleic acid of claim 22 or the plurality of nucleic acids of claim 23.

26. The cell of claim 25, wherein the cell is a yeast cell.

27. The cell of claim 26, wherein the yeast is of the genus Pichia.

28. The cell of claim 26, wherein the yeast is of the genus Saccharomyces.

29. The cell of claim 26, wherein the yeast is of the genus Yarrowia.

30. The cell of claim 25, wherein the cell is a bacterial cell.

31 . The cell of claim 30, wherein the bacteria is of the genus Escherichia.

32. The cell of claim 30, wherein the bacteria is a probiotic bacterium.

33. The cell of claim 32, wherein the probiotic bacteria is selected from the group consisting of the genus Bacillus, the genus Lactobacillus, the genus Bifidobacterium

34. The cell of claim 25, wherein the cell is an insect cell.

35. The cell of claim 34, wherein the insect cell is of the genus Spodoptera.

36. The polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 synthesized in any de novo protein synthesis system

37. The polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 further comprising a vitamin, an amino acid, a dye, an antibiotic, an antiviral, a hormone, an antimicrobial peptide, a steroid, a prebiotic, a probiotic, a bacteriophage, chitin, chitosan, B-1 ,3-glucan, vegetable extracts, peptone, an additive spray, a toxin binder, a yeast extract, a plant extract, sugar, a detoxifying enzyme, an enzyme inhibitor, an essential mineral, carnitine, glucosamine, an essential salt, a preservative, a stabilizer, a pesticide, a herbicide, or water.

38. A method of producing the polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 , comprising (a) incubating a cell of any one of claims 25 to 35 in a medium suitable for secretion of the polypeptide from the cell; and (b) purifying the polypeptide from the medium.

39. The polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 , for use in reducing or preventing a plant-associated bacterial infection in any plant species.

40. Use of the polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 for reducing transmission or preventing transmission of a plant-associated bacterium from a plant species to another plant of the same species or a plant of a different species.

41. Use of the polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 for reducing transmission or preventing transmission of a plant-associated bacterium from a plant species to an insect species.

42. Use of the polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 for reducing transmission or preventing transmission of a plant-associated bacterium from an insect species to a plant species.

43. The use of claim 39 or 40 or 41 or 42, wherein the plant is a grape, olive, almond, citrus, fig, alfalfa, periwinkle, peach, plum, coffee, oleander, pecan, or vaccinium berry plant.

44. The use of claim 39 or 40 or 41 or 42, wherein the insect species is a leafhopper, sharpshooter, or spittlebugs.

45. The use of claim 39 or 40 or 41 or 42, wherein the polypeptide is adapted for inclusion in any part of the plant, introduction to the xylem, introduction to the phloem, provided to the exterior surface (for example, as a spray or submersion), coated on seeds, provided to the medium in which the plant dwells, provided by injection, provided via diffusion, provided via absorption by roots, provided genetically as a genetically- modified plant, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects.

46. The use of claim 39 or 40 or 41 or 42, wherein the polypeptide is adapted for inclusion in any part of the insect, provided by insect feed, provided by insect traps, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects.

47. Use of the polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 for modulating a plant immune system.

48. Use of the polypeptide of any one of claims 1 to 7 or 9 to 21 or the polypeptide complex of any one of claims 8 to 21 to deliver molecules to specifically target a pathogen.